weather, microclimate, canopy density and neighbouring non

Weather, Microclimate, Canopy Density and Neighbouring Non-Host Crop

Impacts on Sclerotinia Stem Rot Disease in Canola

by

Reanne Pernerowski

A Thesis submitted to the faculty of graduate studies of

The University of Manitoba

in partial fulfilment of the requirements for the degree of

MASTER OF SCIENCE

Dept. of Soil Science

University of Manitoba

Winnipeg, Manitoba

Copyright © 2014 by Reanne Pernerowski

ii

ABSTRACT

Pernerowski, Reanne. M.Sc., The University of Manitoba, January 2014. Weather,

microclimate, canopy density and neighbouring non-host field impacts on sclerotinia stem

rot disease in canola. Major Professors; Paul R. Bullock and W.G. Dilantha Fernando.

Sclerotinia stem rot (SSR) disease is one of the most devastating diseases of canola in the

Canadian prairies caused by the fungus Sclerotinia sclerotiorum (Lib.) de Bary. Yield

losses ranging between 5 to 100 percent can be experienced as a result of this disease.

This study evaluated the impacts of weather and microclimate on SSR development in

canola with varying canopy density. Ascospore dispersal and disease incidence were

compared under modified canopy densities and misting regimes to alter microclimate.

The effectiveness of crop rotation and the influence of neighbouring non host crops were

also analyzed in this study. A randomized complete block design was used to compare

values for canopy density, microclimate and disease development under 3 seeding rates

and 3 fertilizer treatments. This design was implemented over 4 site-years, in Winnipeg

and Carman during 2011 and 2012. Weather stations were installed to monitor

environmental conditions at each site and compare these to disease. At each site, a wheat

plot was created to examine ascospore release under a non-host crop to determine the

influence such a crop may have on neighbouring canola fields. Results of this study

showed that peaks in ascospore concentrations occurred simultaneously between

Winnipeg and Carman fields during both years indicating that regional weather

conditions are important for ascospore release. Disease development in canola fields

iii

occurred where adequate precipitation and relative humidity were present prior to

ascospore release and dispersal. A decrease in relative humidity and an increase in

temperature were required for spore release from apothecia. Disease development was

greater in Carman, where relative humidity values overall were higher and temperatures

remained lower compared to those in Winnipeg in 2011 and 2012. Ascospore release did

occur under the wheat canopy and ascospores were dispersed to a distance of at least 7

meters from the plot.

iv

ACKNOWLEDGMENTS

I would like to acknowledge all the individuals and organizations that have helped

throughout the progress of this study. This project would not have been possible without

the funding provided by The Canola Council of Canada and the University of Manitoba.

My advisors, Drs. Paul Bullock and W.G. Dilantha Fernando initiated the project and

guided me throughout the field, lab, data analysis and writing process. I‟d also like to

thank my committee members Annemieke Farenhorst and Jeannie Gilbert for their

support and guidance throughout this project.

Thanks to Bob Terhorst (University of Manitoba Research Station at The Point in

Winnipeg) and Alvin Iverson (University of Manitoba Research Station in Carman) for

providing me with access to research stations for the use of field plots. Bob Terhorst and

Alvin Iverson have also graciously helped with the preparation and takedown of my

fields. Dr. Guo Xiaowei has provided me with assistance in setting up the rotorods and

provided me with guidance on the microscope work in the lab. Without the sclerotia and

spores obtained and given to me for inoculation in my fields and for laboratory testing

and PCR analysis this project would not have been possible, for this I would also like to

thank Dr. Khalid Rashid.

v

I‟d like to give special thanks to those who have helped me in the field and laboratory,

including Paula Parks (Technician, Plant Science), Tim Stem (Technician Soil Science),

Bo Pan (Technician, Soil Science), Besrat Demoz (Technician Plant Science) as well as

the summer students (Michael Chiasson, Jennifer Ellis, Brenleigh Ramm, Michelle

Lacroix, Todd Pernerowski, Ives Nikiema and Samantha Erichsen).

Additional assistance was provided by administrative staff, professors and students at the

University of Manitoba‟s soil science department including Terri Ramm and Lynda

Closson. Francis Zvomuya kindly helped me with SAS (Statistical Analysis Software)

and some data analysis. Michael Cardillo also helped with field setup and laboratory

work.

Lastly, I‟d like to especially thank my current employer (DL Seeds), family and friends

for their patience, encouragement and guidance throughout the writing process.

6

Contents ABSTRACT .................................................................................................................... ii

ACKNOWLEDGMENTS .............................................................................................. iv

LIST OF TABLES........................................................................................................... 8

LIST OF FIGURES ....................................................................................................... 10

1. INTRODUCTION ................................................................................................. 13

1.1 Importance of Canola in Canada ...................................................................... 13

1.2 Sclerotinia Infection ........................................................................................ 14

1.3 Sclerotinia Control........................................................................................... 16

1.4 Weather Impacts on Sclerotinia ....................................................................... 20

1.5 Sclerotinia Disease Models ................................................................................ 1

1.6 Research Objectives .......................................................................................... 3

1.7 References ......................................................................................................... 4

2. CANOPY DENSITY, MICROCLIMATE AND WEATHER IMPACTS ON

SCLEROTINIA STEM ROT DISEASE IN CANOLA .................................................... 8

2.1 Abstract ............................................................................................................. 8

2.2. Introduction ..................................................................................................... 10

2.3 Methods .......................................................................................................... 16

2.3.1 Study Sites ............................................................................................... 16

2.3.2 Field Preparation ...................................................................................... 16

2.3.3 Inoculation ............................................................................................... 21

2.3.4 Microclimate Monitoring .......................................................................... 22

2.3.5 Weather Station Monitoring ..................................................................... 25

2.3.6 Ascospore Dispersal ................................................................................. 26

2.3.7 Petal Sampling ......................................................................................... 30

2.3.8 Misting ..................................................................................................... 33

2.3.9 Crop Development ................................................................................... 34

2.4.1 Disease Incidence ..................................................................................... 37

2.4.2 Data Analysis ........................................................................................... 38

2.4 Results ............................................................................................................. 40

2.4.1 Summary of Results ................................................................................. 40

2.4.2 Weather Impacts on Ascospore Concentrations ........................................ 45

2.4.3 Weather Impacts on Disease Incidence ..................................................... 56

2.4.4 Misting and Seeding Rate Treatment Effects on the Canola Canopy ......... 58

7

2.5 Discussion ....................................................................................................... 80

2.5.1 Weather and Disease Development ........................................................... 80

2.5.2 Effect of Microclimate on Disease Development ...................................... 90

2.6 Conclusion ...................................................................................................... 96

2.7 References ....................................................................................................... 98

3. INFLUENCE OF NON-HOST CROPS IN NEIGHBOURING FIELDS ON

SCLEROTINIA STEM ROT DISEASE ...................................................................... 103

3.1 Abstract ......................................................................................................... 103

3.2 Introduction ................................................................................................... 104

3.3 Methods ........................................................................................................ 107

3.3.1 Study Sites and Field Layout .................................................................. 107

3.3.2 Field Preparation .................................................................................... 108

3.3.3 Microclimate Monitoring ........................................................................ 109

3.3.4 Ascospore Dispersal ............................................................................... 110

3.3.5 Misting ................................................................................................... 111

3.3.6 Growing Season ..................................................................................... 112

3.3.7 Data Analysis ......................................................................................... 113

3.4 Results ........................................................................................................... 114

3.4.1 Weather and Microclimate Impacts on Ascospore Release in the Wheat

Plots 114

3.4.2 Impact of Wind on Ascospore Dispersal from the Wheat Plots ............... 120

3.43 Dispersal Comparisons among Wheat and Canola Plots .......................... 124

3.5 Discussion ..................................................................................................... 127

3.6 Conclusion .................................................................................................... 136

3.7 References ..................................................................................................... 137

4. OVERALL SYNTHESIS ..................................................................................... 139

4.1 References ..................................................................................................... 144

Appendix A ................................................................................................................. 145

8

LIST OF TABLES

Table 2.1 Seeding and fertilizer treatment rates for Winnipeg and Carman field

sites in 2011 and 2012. ................................................................................................18

Table 2.2. Growth stages of Canola and date at which these stages were initially

observed at Carman and in Winnipeg. Note that Winnipeg and Carman were

seeded on May 26th

and June 8th respectively in 2011. In 2012 Carman and

Winnipeg were seeded on May 23rd

and June 6th

respectively. ...................................34

Table 2.3. Dates for growing season, sampling period, ripening, early and late

bloom stages during all four site years. ........................................................................42

Table 2.4. Summary of measurements for canopy density (LAI, plant counts),

weather parameters, ascospore concentrations and percentage of infection during

the growing season, sampling period, ripening, early bloom and late bloom for all

four site years. .............................................................................................................44

Table 2.5. Significance levels from analysis of variance on the effects of seeding

rate on leaf area index (LAI) and plant counts from Winnipeg and Carman in 2011

and 2012 under natural weather conditions (non-misted plots). Words in brackets

indicate which selection (year or location) is higher. ....................................................61


rate and misting on leaf area index (LAI) and plant counts in Winnipeg and

Carman in 2011. Words in brackets indicate which selection (location, misting) is

higher. .........................................................................................................................61


rate on microclimatic parameters including relative humidity (RH) in %,

temperature (T) in °C, growing degree days (GDD) in days and leaf wetness (LW)

from Winnipeg and Carman in 2011 and 2012 under natural weather conditions

(non-misted plots) during the flowering period. Words in brackets indicate which

selection (year or location) is higher. ...........................................................................63

Table 2.8. Levels of significance from analysis of variance on the effects of

seeding rate treatment and misting on microclimate parameters in Winnipeg and

Carman in 2011 during the flowering period. Words in brackets indicate which

selection \misting or location) is higher. .......................................................................64

Table 2.9. Summary of whole plot mean ascospore concentrations (ascospores/m3),

disease incidences (%) and microclimate parameters. 2011 misted and non-misted

mean ascospore concentrations, disease incidences and microclimatic parameters

are listed. Data was used only for days where all values are present and for over

lapping Winnipeg and Carman days only. ....................................................................66


rate on average daily mean ascospore concentrations and disease incidence from

9

Winnipeg and Carman in 2011 and 2012 under natural weather conditions (non-

misted plots). Words in brackets indicate which selection (year or location) is

higher. The natural log was taken for all disease incidence values after they were

added by 2 to obtain a normal distribution. ..................................................................69


rate and misting on average daily mean ascospore concentrations (Spore

Concentration) and disease incidence (DI) in Winnipeg and Carman in 2011.

Words in brackets indicate which selection (location, misting) is higher. The

natural log was taken for all disease incidence values after they were added by 2 to

obtain a normal distribution. ........................................................................................69

Table 2.12. Petal counts containing ascospores within each plot at various bloom

stages. Non listed plots contained 0 ascospore colonized petals. ...................................79

Table 3.1 Start and end dates for specified time periods during the growing season. . 112

Table 3.2. Rotorod sampling period and growing season weather parameters above

the canopy for Winnipeg and Carman in 2011 and 2012. Maximum and minimum

temperatures are indicated in brackets beside mean temperature values...................... 116

Table 3.3. Microclimate parameters within the wheat strips in Winnipeg and

Carman in 2011 and 2012 during the rotorod sampling period, from 4th

leaf to

flowering stage and during the flowering stage for canola. Calculated values are

obtained from the average of two microclimate stations placed within the wheat

strips. ......................................................................................................................... 116

Table 3.4. Measured values for ascospore concentrations and microclimate

parameters in Winnipeg and Carman in 2011 for misted and non-misted wheat

field portions during the sampling period. .................................................................. 119

Table 3.5. Parameters obtained by an exponential model applied to mean spore

concentrations at 3 meters and 7 meters from an inoculum source in Winnipeg.

50% reduction and 75% reduction indicate the distance at which a 50 percent and

75 percent reduction in average mean spore concentration would occur. .................... 123

Table 3.6. Average daily mean ascospore concentrations (ascospores/m3) and

disease incidence averages within canola plots at 30-40, 20-30 and 10-20 metres

from inoculated wheat source in 2011 and 2012 Carman plots. .................................. 124

Table 3.7. Whole plot means for average daily mean ascsopore concentrations in

wheat and canola plots and percentages of infection obtained at harvest in canola

plots at Winnipeg and Carman in 2011 and 2012. Ascospore concentration data

used was taken only for days in which all values were present in both wheat and

canola during a specified site year over the sampling period. ..................................... 125

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LIST OF FIGURES

Figure 2.1 Canola field set up for Winnipeg and Carman in 2011 (left) and 2012 (right).

High, medium and low seeding and fertilizer rates are represented by dark grey, light grey

and white shades within canola plots..............................................................................20

Figure 2.2. Diagram of canola growth stages in Winnipeg and Carman in 2011. ...........34

Figure 2.3. Diagram of canola growth stages in Winnipeg and Carman sites in 2012. ....35

Figure 2.4. Comparison of average daily mean ascospore concentrations over the

sampling period in misted (dashed lines) and non-misted (solid lines) plots in Winnipeg

(green lines) and Carman (blue lines) in A) 2011 and B) 2012. ......................................46

Figure 2.5. Seasonal comparisons between Winnipeg (green lines) and Carman (blue

lines) in I) 2011 and II) 2012 among A) precipitation (mm) (bars), relative humidity (%)

(solid lines), B) temperature (°C) (solid lines), solar radiation (kw/m2) (dashed lines) and

C) wind speeds (m/s) (solid lines). .................................................................................47

Figure 2.6. Cumulative mean daily ascospore concentrations (red) and total daily

precipitation (purple) from the 1st to the 24

th day of flowering in Carman 2011 (dotted

line), Winnipeg 2012 (solid line) and Carman 2012 (dashed line). .................................49

Figure 2.7. Mean daily ascospore concentrations in ascospores per m3 in A) Carman 2011

in misted (black lines) and non-misted (grey lines) plots B) Winnipeg 2011 in misted

(black lines) and non-misted (grey lines) plots C) Carman 2012 in non-misted (grey

lines) plots and D) Winnipeg 2012 in non-misted (grey lines) plots plotted with total daily

rainfall in mm (black bars) in A) Carman 2011 B) Winnipeg 2011 C) Carman 2012 and

D) Winnipeg 2012. The flowering period is the time period between the * symbols. ......50

Figure 2.8. Mean daily ascospore concentrations in ascospores per m3 (red lines) plotted

with average daily I) temperatures in degrees celsius (purple lines) and II) relative

humidity in percentage (black lines) for A) Carman 2011 B) Winnipeg 2011 C) Carman

2012 and D) Winnipeg 2012. The flowering period is indicated by the area within the *

symbols. ........................................................................................................................53

Figure 2.9. Mean daily ascospore concentrations in ascospores per m3 (red lines) plotted

with I) mean daily wind speed (purple lines) and II) total daily solar radiation (black

lines) for A) Carman 2011 B) Winnipeg 2011 C) Carman 2012 and D) Winnipeg 2012. 54

Figure 2.10. Average of daily mean ascospore concentrations for non-misted plots in

Carman 2011, Winnipeg 2011, Carman 2012 and Winnipeg 2012 occurring within

specified intervals for factors including A) mean daily air temperature (°C), B) mean

daily relative humidity (%), C) minimum daily relative humidity (%), D) daily decrease

in relative humidity from 8am to 2pm (%) E) total daily precipitation (mm) and F) mean

daily wind speed (m/s). ..................................................................................................55

Figure 2.11. Percent infection counts during harvest (black diamond) and after harvest

(grey diamond) in A) Carman 2011, B) Winnipeg 2011, C) Carman 2012 and D)

11

Winnipeg 2012. Weather parameters include total daily precipitation in mm (black bars),

temperature in °C (grey lines) and relative humidity in % (black lines). .........................57

Figure 2.12: Leaf area index values taken from plots 1-18 throughout the A) 2011

growing season in Winnipeg (plots 10-18 are misted) B) 2011 growing season in Carman

(plots 1-9 are misted) C) 2012 growing season in Winnipeg and D) 2012 growing season

in Carman. Blue, green and dotted yellow lines represent plots seeded at high, medium

and low seeding rates respectively. ................................................................................60

Figure 2.13. Accumulated ascospore concentrations (ascospores/m3) in high (blue),

medium (green) and low (yellow) plots in Carman 2011, Winnipeg 2012 and Carman

2012. .............................................................................................................................70

Figure 2.14. Relationship between sclerotinia stem rot disease incidence (%) and a) LAI;

b) plant density in Winnipeg 2011 (black line), Carman 2011 (grey line), Winnipeg 2012

(dotted black line) and Carman 2012 (dotted grey line). .................................................72

Figure 2.15. Relationship between ascospore concentration (ascospores/m3) and a) LAI;

b) plant density in Winnipeg 2011 (black line), Carman 2011 (grey line), Winnipeg 2012

(dotted black line) and Carman 2012 (dotted grey line). .................................................73

Figure 2.16. Comparisons of A) ascospore concentrations (ascospores/m3) and overall

microclimatic averages in high, medium and low plots for B) daily air temperature (˚C),

C) daily relative humidity (%), D) daily soil temperature (˚C) and E) daily leaf wetness in

Carman, 2011. ...............................................................................................................75




Winnipeg, 2011. ............................................................................................................76




Carman, 2012. ...............................................................................................................77




Winnipeg, 2012. ............................................................................................................78

Figure 3.1 Comparison of daily microclimate factors such as B) air temperature in ˚C

(black line), and soil temperature in ˚C (grey line) and C) leaf wetness (black line) and

relative humidity in percentage (grey line) to A) daily mean ascospore concentrations

(ascospore/m3) in I) Carman and II) Winnipeg in 2011. Red lines indicate the beginning

and the end of the flowering period. ............................................................................. 117

Figure 3.2. Comparison of daily microclimate factors such as B) air temperature in ˚C

(black line), and soil temperature in ˚C (grey line) and C) leaf wetness (black line) and

relative humidity in percentage (grey line) to A) daily mean ascospore concentrations

(ascospore/m3) in I) Carman and II) Winnipeg in 2012. Red lines indicate the beginning

and the end of the flowering period. ............................................................................. 118

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Figure 3.3. Design of bare and wheat field portions for A) Carman 2011 B) Winnipeg

2011 C) Carman 2012 and D) Winnipeg 2012. Circles represent rotorod locations with

average daily mean ascospore concentrations within them for the sampling periods at

each field at both locations. Black arrows in the bottom right corners represent dominant

wind directions during the sampling periods. Rotorods were placed at 3 and 7 metres

from inoculated wheat strips in bare soil. Image is not to scale. .................................... 121

Figure 3.4. Average daily mean ascospore concentrations in wheat (black line) and canola

(grey line) throughout the sampling period in A) Carman 2011, B) Winnipeg 2011, C)

Carman 2012 and D) Winnipeg 2012. .......................................................................... 126

Figure A.1. Canola plots containing 1 microclimate station and 1 rotorod in each

plot during flowering in Carman, Manitoba (2012) .....…………………………....…...168

Figure A.2. Wheat strip located adjacent to the canola plots with rotorods and

microclimate stations in Winnipeg, Manitoba (2011)……….………..…………….…168

Figure A.3. Canola plots in Winnipeg (2011) with microclimate stations and rotorods

during the leaf stage……………...…………………………………………………..…169

Figure A.4. Rotorod spore sampling device……………………………………...……..170

Figure A.5. Hobo microclimate station…………………………………………...…….171

Figure A.6. Sclerotinia stem rot disease life cycle including management practices in blue

indicated by the numbers 1 through 7 and environmental conditions impacting disease

development in red indicated by letters A through F……………………….……..……172

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1. INTRODUCTION

1.1 Importance of Canola in Canada

Canola is a highly profitable crop grown in Canada, contributing $19.3 billion to

the Canadian economy each year and generating one fourth of all farm cash receipts

through sales and exports to the US and other countries worldwide (Canola Council of

Canada, 2011); yield losses in this crop can be very costly to the Canadian economy.

Increasing demands for canola encourage the need for higher yielding varieties and

increased acreage. Various products are manufactured from harvested canola seed

including oil for human consumption (44% of harvested canola) as well as meal for

livestock (56% of harvested canola) and recently for biodiesel (Canola Council of

Canada, 2011). Canola is mostly grown in Alberta, Saskatchewan and Manitoba, although

a substantial amount of crop is also grown in British Columbia, Ontario and Quebec

(Canola Council of Canada, 2013). Canola is particularly important in Manitoba and due

to increased seeded acreage, the frequency of canola in rotations is increasing (McLaren

et al., 2012). Specifically in Manitoba, 3.5 million acres of canola were seeded in 2012 to

meet increasing demand for the crop (McLaren et al., 2012).

Several fungal diseases posing major concerns in canola are prevalent in Canada,

including sclerotinia stem rot (SSR), blackleg, alternaria blackspot, seedling disease

complex and root rot complex (Canola Council of Canada, 2011). In Western Canada,

SSR is one of the most serious diseases in canola. It is caused by the fungus Sclerotinia

14

sclerotiorum (Lib.) de Bary and has been present in Manitoba and Saskatchewan for the

past 25 years (Manitoba Agriculture, 2013). Sclerotinia has been identified worldwide,

however it is more prevalent in temperate and subtropical regions with cool and wet

climates (Purdy 1979). Sclerotinia stem rot is capable of affecting over 400 host crops

worldwide including dry beans, field peas, lentils, mustard, potatoes, sunflower and

canola (Manitoba Agriculture, 2013). Sclerotinia has been known to reduce yields in

canola by an average of 0.4 to 0.5 times the percentage of infection, causing losses

ranging from 5-100% in the Canadian prairies (Manitoba Agriculture, 2013). Sclerotinia

has been the most prevalent disease in canola in surveys conducted annually to determine

impacts to canola crops (McLaren et al., 2012).

1.2 Sclerotinia Infection

Sclerotinia sclerotiorum and Sclerotinia minor are the two sclerotinia species

present in Canada, with S. sclerotiorum capable of infecting 408 species of crops (Boland

and Hall 1988) and S. minor capable of infecting 94 species (Melzer et al., 1997).

Sclerotinia species can infect plants by either carpogenic germination resulting in the

production of ascospores infecting above ground tissues (Sclerotinia sclerotiorum) or

myceliogenic germination infecting the roots of host plants (Sclerotinia minor) (Bardin

and Huang, 2001). Ascospores are the primary source of inoculum; mycelial infections

are rare in nature (Abawi and Grogan, 1979). Myceliogenic germination causes plant wilt

in various plant species (Huang and Erikson, 2002). Diseases caused by carpogenic

germination include white mold and pod rot of bean, stem blight and leaf blight of canola,

pod rot of peas, head rot of sunflower, lettuce drop and blossom blight of alfalfa (Huang

and Erikson, 2002; Fernando et al. 2004). The mechanism by which the pathogen infects

15

plants depends on the host crop and environmental conditions at that particular location.

Canola crops in Western Canada are typically only infected by ascospores of Sclerotinia

sclerotiorum (Bardin and Huang, 2001).

The infection process for S. sclerotiorum begins in the spring or summer (Figure

A.1). Sclerotinia sclerotiorum begins its life cycle as dormant sclerotia; irregularly

shaped fungal structures capable of surviving in the soil for prolonged periods of time,

generally up to four years (Manitoba Agriculture, 2013). In Manitoba, sclerotia are

present in the soil throughout the winter when they are exposed to low temperatures

allowing for conditioning of the fungal body for future stimulation or acceleration of

apothecia production (Clarkson et al., 2007). During the spring or early in the summer,

the sclerotia germinate through asexual reproduction and produce apothecia, which

appear as mushroom-like structures at the soil surface (Manitoba Agriculture, 2010).

Apothecia produce asci from which airborne ascospores are produced asexually and are

forced into the atmosphere (Harthill and Underhill, 1976). In order for germination and

infection to occur on the plant, the ascospores require a readily available food source by

initially landing on canola flowers, fallen petals and pollen located on the stems and

leaves (Manitoba Agriculture, 2010). Infection occurs when the petals fall on the stems

and leaves of the canola plant (Government of Saskatchewan, 2009). Once deposited onto

the leaf, ascospores are capable of surviving for up to 12 days depending on their position

in the canopy and environmental conditions (Caesar and Pearson, 1983). Once

established, mycelium releases oxalic acid and acidic enzymes into the plant, disrupting

vascular tissue and killing the plant (Boland and Hall, 1988). Secondary spread from

16

diseased to healthy tissue is possible through direct contact (Huang and Hoes, 1980).

Sclerotia are newly formed in and on infected tissues (Huang and Erikson, 2002).

Initial symptoms of sclerotinia stem rot disease in canola appear as water-soaked

spots followed by pale grey to white lesions on stems. Infection then spreads to branches

and pods several weeks after the onset of flowering (Manitoba Agriculture, 2014).

Infected stems appear bleached and hard black fungal bodies are often found within

shredded stems, branches or pods (Manitoba Agriculture, 2014). Canola infected with

sclerotinia will ripen prematurely, wilt and lodge much more easily (Manitoba

Agriculture, 2010). Individual plants can be rated for disease severity using a 0-5 scale,

where 0 indicates no disease and 5 indicates a plant infected completely. Fields and plots

can also be rated for percentage of infection by counting the number of infected plants

over the total number of plants. This can be done in small sample units and extrapolated

over entire plots or fields.

1.3 Sclerotinia Control

Management of sclerotinia stem rot is increasingly important due to the severe

economic losses experienced by canola crops exposed to the disease. Very few

commercially available canola crop varieties are resistant to the pathogen and breeding is

difficult due to the need for multiple genes (Fuller et al., 1984). Canola varieties that

resist lodging or reduce canopy density are beneficial in sclerotinia control (Canola

Council of Canada, 2011). Canola varieties with some physiological tolerance have been

recently introduced by DuPont Pioneer with 45S54 marketed in 2013, and by Bayer Crop

Science with their sclerotinia tolerant InVigor variety that will be launched in 2014.

17

Sclerotinia resistant varieties are only effective in reducing severity and do not always

eliminate the need for fungicide use.

1.3.1 Fungicides:

Sclerotinia stem rot disease is primarily controlled by the use of fungicides,

although this method is not always recommended due to the costs associated with the

purchase of fungicides and sporadic nature of the disease making it difficult to effectively

time fungicide applications (Bradley et al., 2006). Current registered fungicides include

Astound (cyprodinil and fludioxonil), Lance (boscalid), Overall (iprodione), Proline

(prothioconaole), Quadris (azoxystrobin) and Rovral Flo (iprodione) (Manitoba

Agriculture, 2013). Generally, chemical fungicides are applied at 20-50 percent bloom

stage before symptoms are visible (Manitoba Agriculture, 2013). Once symptoms are

present, fungicide application will not help in the management of the disease (Manitoba

Agriculture, 2013).

Although the main method to reduce infection of SSR is through fungicide

application, other control methods have been proven to reduce infection as well. Seeds

infected by S. sclerotiorum generally fail to germinate or seedlings die upon germination

then become rotted with sclerotia forming in the seed (Fernando et al., 2004). The fungus

can be removed from the infected seed through the use of seed treatment.

1.3.2 Biological Control:

Due to environmental concerns associated with the use of chemicals in agricultural

crops, biological control is an attractive alternative. This can be accomplished through the

presence and addition of biological control agents including fungal and bacterial

antagonists. These agents are capable of reducing sclerotia populations before the

formation of apothecia and release of ascospores (Fernando et al., 2004). Coniothyrium

18

minitans is an example of a widely studied fungal antagonist capable of reducing airborne

ascospore infections (Fernando et al., 2004). C. minitans is applied prior to planting a

spring crop in a solution called Contans (Manitoba Agriculture, 2013). Bacterial

antagonists include Bacillus and Pseudomonas species, however little research has been

conducted on the use of these species in SSR management. Organic and inorganic soil

ammendments can also be added to the soil in order to promote the growth of soil-borne

micro-organisms to reduce sclerotia germination and suppress S. sclerotiorum

germination (Bardin and Huang, 2001).

1.3.3 Cultural Methods:

Cultural methods are also used instead of or in conjunction with chemical

applications. These methods include tillage, flooding, reduced irrigation, rotation with

non-host crops and biological control agents (Bardin and Huang, 2001). The

development of environmentally-friendly management strategies is required to effectively

control the infection of sclerotinia on canola. This can be more easily addressed through

understanding the causes of the disease and its rate of infection and severity as a response

to microclimate and standard weather conditions. Management can be further enhanced

by early identification of crops at risk and proper timing of fungicide applications.

Microclimate modifications can also be managed to create conditions unfavourable to

disease development via seeding rate and row spacing modifications.

Burial depth is an important factor affecting the germination and viability of

sclerotia in field settings (Duncan et al., 2005). Stipes can generally grow no longer than

3 cm in the field; therefore the only functional sclerotia are found in the top 2 to 3cm of

the soil column (Abawi and Grogan, 1979; Wu and Subbarao, 2008). Tillage, as a method

to control sclerotial germination may also be effective as a management practice. Tilling

19

sclerotia-containing soils effectively buries the sclerotia deeper into the soil profile,

reducing their ability to receive sunlight and germinate (Wu and Subbarao, 2008);

however resurfacing of the fungal bodies is possible. Other studies have shown that a zero

tillage method could be beneficial in reducing future sclerotia inoculum levels in the soil.

Mycoparasites are present in the top soil and are capable of deteriorating the fungal

bodies (Tu, 1989). Survival is therefore prolonged in tilled soil where mycoparasite

numbers may be reduced.

Crop rotation strategies are implemented to reduce sclerotia populations in fields and

alleviate disease history. The Canola Council of Canada (2011) recommends at least 1

year between canola crops; 2 to 3 years are preferred. Although this strategy is widely

used to increase canola yields and reduce pressures from pests and disease (Canola

Council of Canada, 2013), it may not always benefit the farmer. Limitations for rotating

with non-susceptible crops include; the limited availability of non-host crops (Manitoba

Agriculture, 2010), duration of rotations required to deplete sclerotia levels (4 years)

(Bradley et al., 2006) and disease pressures from neighbouring fields. Williams and

Stelfox (1979) found that sclerotia germination increased under consecutive years of

rapeseed (host) crops. Under non-host crops, germination rates remained the same each

year; therefore inoculum depletion did not occur (Schwartz and Steadman, 1978;

Williams and Stelfox, 1979). According to Williams and Stelfox (1979), ascospores are

capable of travelling a distance of 150 m and up to 7m high from the apothecia source,

Additional study on sclerotia longevity and depletion rates in non-host crops is required.

Ascospore release and dispersal is more defined under specified environmental conditions

in a wheat (non-host) crop in chapter 3.

20

1.4 Weather Impacts on Sclerotinia

Microclimatic conditions are influenced by plant and canopy density which is

altered through the modification of seeding rates as well as row spacing. Jurke and

Fernando‟s (2008) study demonstrated that plant density does affect sclerotinia stem rot

incidence in canola. They compared several seeding rates over three years with resulting

sclerotinia disease incidence. Dense canopies have been shown to create conditions where

relative humidity is higher with longer periods of leaf wetness (Turkington, 1991) and

temperatures tend to be lower (Blad et al., 1978). With certain cultivars, lodging also

became more apparent where higher density crops were situated creating microclimatic

conditions where development of sclerotinia stem rot was increased (Jurke and Fernando,

2008). Where lodging is observed, there is generally an increase in soil surface moisture

under the canopy and there is also more contact between plants both creating conditions

conducive for SSR development and allowing for the disease to spread among plants

(Jurke and Fernando, 2008). A study on soybean also demonstrated that wider row

spacing decreased the incidence of disease (Steadman et al., 1973). Additional studies

focusing on the effects of canopy density on microclimate is essential for a better

understanding of the impact of canopy density on disease.

Researchers have attempted to determine critical conditions required for sclerotia

germination, ascospore release and infection (Table 1.1). Soil moisture is the most

common limiting factor on germination (Matheroon and Porchas, 2005; Mila and Yang,

2008). Germination increases with additional moisture supply during prolonged periods

of time and are destroyed under saturated conditions. Soil moisture may not be required

21

for sclerotia germination in dry regions such as Arizona (Matheroon and Porchas, 2005).

With respect to temperature, optimum sclerotia germination rates are variable depending

on the geographical region from which sclerotia are collected. Temperature requirements

also vary depending on the presence of light and moisture. Precipitation and irrigation

favour sclerotia germination by reducing temperatures and increasing moisture supply to

the canopy (Gugel and Morall, 1986; Teo et al., 1989). Light, temperature and moisture

regimes influence the appearance of stipes (Sun and Yang, 2000). Researchers have found

that ascospore release has occurred under both light and dark conditions (Clarkson et al.,

2003), contrary to the discovery by Harthill (1980) that light was involved in the process.

This finding leads to the assumption that other factors including temperature or relative

humidity are responsible for daytime release of spores (Clarkson et al., 2003). Qandah

and Mendoza (2011) attempted to validate several findings by relating environmental

factors to the daily and seasonal release and dispersal patterns of ascospores into the

atmosphere. Their three year study (2005 to 2007) during canola flowering suggests that

temperature and relative humidity have a significant impact on ascospore release. Hourly

observations showed that ascospore dispersal occurs in a single event lasting between

four to six hours no more than once daily, either at night or during the day depending on

the year. Although these weather variables have been extensively studied, McCartney and

Lacey (1991) have shown that ascospore concentrations in the air were more closely

related to the number of apothecia produced than to weather variables. Spore survival

may also be a limiting factor in the infection by ascospores on various crops under

conditions of high temperature and relative humidity (Clarkson et al., 2003). Precipitation

and irrigation are responsible for infection as heavy irrigation practices increased disease

severity in a study on dry edible beans (Weiss et al., 1980). Spring rainfall also leads to

22

ideal conditions for disease development, favouring sclerotia germination (Kirkegaard et

al., 2006). Plant wetness is also required for infection of ascospores on nutrient-

containing tissues such as petals for further development of stem rot of canola (Bardin

and Huang, 2001). A disease prediction model in canola that can relate weather

conditions such as rainfall to soil moisture content and leaf wetness would create a better

understanding of the development of sclerotinia stem rot and also allow for better

prediction of disease occurrence (Table 1.1).

1

Table 1.1 Factors influencing disease development (including sclerotia germination, ascospore release and infection) of sclerotinia stem

rot disease according to past research.

Factor Value Influence on disease Location of experiment Reference Sclerotia Germination Temperature, soil moisture

15-40°C , ≥ 0.02 Mpa decrease sclerotia germination with increasing temperature in wet soil

growth room (Arizona) Matheroon and Porchas, 2005

Temperature 25°C Highest apothecia production growth room (Iowa) Sun and Yang, 2000

Soil temperature 20°C and fluctuating temperature similar effects on sclerotia germination growth room (Iowa) Mila and Yang, 2008

15°C highest germination rates from in sclerotia from california

growth room (California) Wu and Subbarao, 2008

reduction by 15°C favourable for sclerotia germination Field (Saskatchewan) Teo et al., 1989

Soil moisture at or above -0.4 MPa to -0.5 MPa above these levels sclerotia germination is increased

growth room (Saskatchewan); growthroom (Ontario)

Teo and Morall, 1985a; Boland and

Hall, 1987

0 Mpa (saturation) sclerotia rot and are destroyed growth room (Saskatchewan) Teo and Morall, 1985b

continual -0.001 Mpa favours sclerotia germination over fluctuating moisture

growth chamber (Iowa) Mila and Yang, 2008

10 days moist soil sclerotia germination and apothecia production Manitoba Manitoba Agriculture, 2013

increased soil moisture increase in sclerotia germination growth room (California) Wu and Subbarao, 2008

Precipitation 10 mm appearance of appothecia 3-7 days after precipitation

Field (Saskatchewan) Gugel and Morall, 1986

Light, temperature and soil moisture

120 - 190 mol/m/s, 20°C to 25°C require saturated soil for production of short, thick stipes

growth room (Iowa) Sun and Yang, 2000

80 - 120 mol/m/s, 6-12°C require partially (50%) saturated soil for production of long thin stipes

growth room (Iowa) Sun and Yang, 2000

Ascospore Release and Dispersal

Light light, dark light involved in ascospore release lab (New Zealand) Harthill, 1980

spores released in light and dark conditions lab (Worcester, UK) Clarkson et al., 2003

light, temperatureand moisture

high light, high temperature, high relative humidity

large daytime ascospore release during wet years at increasing temperaute and decreasing relative humidity, following a period of high relative humidity

field (North Dakota); United Kingdom Qandah and Mendoza, 2011;

McCartney and Lacey, 1991

no light small ascospore release during dry years at night field (North Dakota) Qandah and Mendoza, 2011

temperature, relative humidity

high temperature, high relative humidity reduction in spore survival lab (Worcester, UK) Clarkson et al., 2003

Infection

Precipitation 5.5 cm every 10 days versus 5.5 cm every 5 days (irrigation)

increased disease severity Weiss et al., 1980

550 - 650 mm rain high levels of sclerotinia stem rot incidence Field (Australia) Kirkegaard et al., 2006

1

1.5 Sclerotinia Disease Models

Models focusing solely on relationships between S. sclerotiorum development

stages have been created. Disease development originates with sclerotia germination

followed by ascospore dispersal and subsequent petal infestation, followed by disease

occurrence as fallen petals land on canola leaves and stems. Gugel and Morall (1986)

have attempted to relate sclerotia germination and petal infestation to disease incidence.

A positive relationship exists between sclerotia germination and disease incidence in

large scale sampling regimes (Gugel and Morall, 1986). Better relationships were found

between petal infestations and disease incidence where petal samples were obtained in

early bloom stages; petal infestations at this stage have the best opportunity to cause

disease because they have a longer period for establishment on the main stem (Gugel and

Morall, 1986). Although petal sampling is a simple and cost effective way to assess

disease risk, monitoring ascospore dispersal is beneficial in assessing disease risk prior to

flowering and ascospore colonization; however this method is not cost effective for

farmers due to the price of spore capturing devices. The simplest and most effective way

to assess risk prior to flowering is to relate disease development to weather conditions.

Researchers have attempted to develop models relating microclimatic and weather

variables to disease incidence. The purpose of the model would be to inform farmers on

the need for and the timing of fungicide application for more cost-effective sclerotinia

control. Risk point systems and disease prediction models were created by Twengstrom et

al. (1998) and Bom and Boland (2000) respectively to provide information on the need

for spraying. They also suggested and modeled factors responsible for disease incidence

2

such as rainfall, soil moisture, canopy density, crop height, growth stage and apothecia

counts (Bom and Boland, 2000). This shows that in addition to the presence of pathogen

and host crop, a favorable environment is necessary for disease to occur. Since the

environment plays a key role in completing the disease cycle, more studies are required to

better quantify the environmental factors that cause sclerotinia stem rot disease in canola.

In doing so, disease prediction will also be enhanced, which, in turn, will facilitate

management decisions about both if and when fungicide application can most effectively

control the disease.

Evaluation of potential disease risk can be accomplished through the use of

various methods; however some are more practical and economical than others. In fields

with unknown history of disease, monitoring of germinated sclerotia can be useful in

determining inoculum levels present within the field and forecasting disease (Gugel and

Morall, 1986; Boland and Hall, 1988). Apothecia counts do not directly represent

inoculum levels as sclerotia germination may be limited by environmental conditions. In

commercial fields, great efforts are required to complete apothecia counts over large

scales but inoculum in the form of ascospores arising from neighbouring fields is also not

taken into consideration. Spore trapping devices used to monitor atmospheric ascospore

concentrations as an indicator of spore release patterns and to forecast disease are

extremely expensive and require frequent visits to the field and timely analysis to

complete spore counts. Use of petal infestations to evaluate risk of sclerotinia stem rot

was suggested by Gugel and Morall (1986) and Turkington (1991). Monitoring of petal

infestations can be accomplished by collecting petals in the field and plating petals on a

nutrient based agar. For large scale sampling in commercial fields, this method can be

3

quite costly. It takes approximately 7 days to observe and identify S. sclerotiorum

mycelium from a plated petal containing ascospores; which is often too late for chemical

applications. The relationship between petal infestation and disease incidence was often

weak and therefore not a useful indicator in forecasting (Turkington, 1991). Several

studies have proven that microclimate can be a predictor of disease development,

however microclimate stations are costly and difficult to use compared to weather

stations. A link between weather, microclimate and disease development is required to

predict sclerotinia stem rot risks. Weather data is widely available and has the potential to

be of great benefit in determining the need for chemical applications based on risks

associated with weather.

1.6 Research Objectives

The specific objectives of this study were:

1) To relate weather to sclerotinia stem rot disease and determine the impact of

microclimatic conditions in varying canola canopy densities on S. sclerotiorum

2) To determine the impact of a sclerotinia infected field containing a non-host

wheat crop on sclerotinia disease development and spread by spore dispersal

4

1.7 References

Abawi, G. S. and Grogan, R. G. 1979. Epidemiology of disease caused by Sclerotinia

species. Phytopathology 69:899-904.

Bardin, S.D. and Huang, H.C. 2001. Research on biology and control of Sclerotinia

diseases in Canada. Canadian Journal of Plant Pathology 23: 88-98.

Blad, B.L., Steadman, J.R. and Weiss, A. 1978. Canopy structure and irrigation

influence white mold disease and microclimate of dry edible beans. Phytopathology

68:1431-1437.

Boland, G.J. and Hall, R. 1987. Epidemiology of white mold of white bean in Ontario.

Canadian Journal of Plant Pathology 9:218-224.

Boland,G.J., and Hall, R. 1988. Relationships between the spatial pattern and number of

apothecia of Sclerotinia sclerotiorum and stem rot of soybean. Plant Pathology 37:329-

336.

Bom, M. and Boland, G. L. 2000. Evaluation of disease forecasting variables for

sclerotinia stem rot (Sclerotinia sclerotiorum) of canola. Canadian Journal of Plant

Science 80: 889–898.

Bradley, C.A., Lamey, H.A., Endres, G.J., Henson, R.A., Hanson, B.K., Mckay, K.

R., Halvorson, M., LeGare, D.G. and Porter, P.M. 2006. Efficacy of fungicides for

control of Sclerotinia stem rot of canola. Plant Disease 90:1129-1134.

Caesar. A. J., and Pearson, R. C. 1983. Environmental factors affecting survival of

ascospores of Sclerotinia scerotiorum. Phytopathology 73:l024-1030.

Canola Council of Canada. 2011. Crop Production. Canola Growers Manual. Contents.

Chapter 10C – Diseases. Chapter 10C. Available from: http://www.canola-

council.org/crop-production/canola-grower's-manual-contents/chapter-10c-

diseases/chapter-10c. Cited on September 11, 2012.

Canola Council of Canada. 2013. Canola Encyclopedia. Diseases. Sclerotinia. Accessed

from: http://canolacouncil.org/canola-encyclopedia/disease/sclerotinia/#about-sclerotinia-

stem-rot

Clarkson, J.P., Stavenley, J., Phelps, K., Young, C. S. and Whipps, J. M. 2003. Ascospore release and survival in Sclerotinia sclerotiorum. Mycol. Res. 107 (2): 213-222.

Clarkson, J. P., Phelps, K., Whipps, J. M., Young, C. S., Smith, J. A. and Watling,

M. 2007. Forecasting sclerotinia disease on lettuce: a predictive model for carpogenic

germination of Sclerotinia sclerotiorum sclerotia. Phytopathology 97:621-631.

http://www.canola-council.org/crop-production/canola-grower's-manual-contents/chapter-10c-diseases/chapter-10c



5

Duncan, R.W., W.G Dilantha Fernando, Rashid, K.Y. 2005. Time and burial depth

influencing the viability and bacterial colonization of sclerotia of Sclerotinia

sclerotiorum. Soil biology and biochemistry. 2005, 1-10.

Fernando, W.G.D., Nakkeeran, S., and Zhang, Y. 2004. Ecofriendly methods in

combating Sclerotinia sclerotiorum (Lib.) de Bary. Recent Research Development

Environmental Biology 1:329-347.

Fuller, P., Coyne, D., Steadman, J. 1984. Inheritance of resistance to white mold

disease in a diallel cross of dry beans. Crop Science 24, 929–933.

Government of Saskatchewan. 2009. Crops – Disease – Sclerotinia Stem Rot

Forecasting in Canola – FAQ. Available from

http://www.agriculture.gov.sk.ca/Default.aspx?DN=a8664793-a3e6-4c6d-b36a-

0dda36e735c7: [cited 12 September, 2011].

Gugel, R.K. and Morall, R. A. A. 1986. Inoculum-disease relationships in sclerotinia

stem rot of rapeseed in Saskatchewan. Canadian Journal of Plant Pathology 8: 89-96.

Harthill, W.F.T. and Underhill, A.P. 1976. Puffing of Sclerotinia sclerotiorum and S.

minor. New Zealand Journal of Botany 14: 355-358.

Harthill, W.F.T. 1980. Aerobiology of Sclerotinia sclerotiorum and Botrytis cinerea

spores in New Zealand tobacco crops. New Zealand Journal of Agricultural Research.

23:256-262.

Huang, H.C. and Erikson, R.S. 2002. Overwintering of Coniothyrium minitans, a

mycoparasite of Sclerotinia sclerotiorum on the Canadian praries. Australasian Plant

Pathology 31:291-293

Huang, H.C. and Hoes, J.A. 1980. Importance of plant spacing and sclerotial position to

development of Sclerotinia wilt of sunflower. Plant Disease 64:81–84.

Jurke, C.J., and Fernando, W.G.D. 2008. Effects of seeding rate and plant density on

sclerotinia stem rot incidence in canola. Archives of Phytopathology and Plant Protection

41(2): 142-155.

Kirkegaard, J.A., Robertson, M.J., Hamblin, P. and Sprague, S.J. 2006. Effect of

blackleg and sclerotinia stem rot on canola yield in the high rainfall zone of southern New

South Wales, Australia. Australian Journal of Agricultural Research 57: 201-212.

Manitoba Agriculture, M.A.F.R.D., 2014. Crops – Diseases – Sclerotinia in Canola.

Available from http://www.gov.mb.ca/agriculture/crops/plant-diseases/sclerotinia-

canola.html: [cited 5 April 2014].

http://www.agriculture.gov.sk.ca/Default.aspx?DN=a8664793-a3e6-4c6d-b36a-0dda36e735c7

http://www.agriculture.gov.sk.ca/Default.aspx?DN=a8664793-a3e6-4c6d-b36a-0dda36e735c7

6

Manitoba Agriculture. 2013. Guide to field crop protection. Plant Disease Control.

Accessible from: http://www.gov.mb.ca/agriculture/crops/insects/guide-to-crop-

protection.html [cited 10 December 2013].

Matheroon, M. E. and Porchas, M. 2005. Influence of soil temperature and moisture on

eruptive germination and viability of sclerotia of Sclerotinia minor and S. sclerotiorum.

Plant Disease 89:50-54.

McCartney, H.A. and Lacey, M.E. 1991. The relationship between the release of

ascospores of Sclerotinia sclerotiorum, infection and disease in sunflower plots in the

United Kingdom. Grana, 30(2): 486-492.

McLaren, D., Kubinec, A., Derksen, H., Bisht, V. and Henderson, T. Manitoba

Canola Disease Survey 2012: Results and Impact. 1Agriculture and Agri-Food Canada,

Brandon, MB R7A 5Y3. Manitoba Agriculture,Food and Rural Initiatives, Carman, MB.

Melzer, M.S., Smith, E.A., and Boland, G.J. 1997. Index of plant hosts of Sclerotinia

minor. Canadian Journal of Plant Pathology 19: 272-280.

Mila, A.L. and Yang, X.B. 2008. Effects of fluctuating soil temperature and water

potential on sclerotia germination and apothecial production of Sclerotinia sclerotiorum.


Purdy, L. H. 1979. Sclerotinia sclerotiorum: history, diseases and symptomatology, host

range, geographic distribution, and impact. Phytopathology 69:875-880.

Qandah, I.S. and del Rio Mendoza, L.E. 2011. Temporal dispersal patterns of

Sclerotinia sclerotiorum ascospores during canola flowering. Canadian Journal of Plant

Pathology 33(2): 159-167.

Schwartz, H. F., and J. R. Steadman. 1978. Factors affecting sclerotium populations of,

and apothecium production by Sclerotinia sclerotiorum. Phytopathology 68: 383-388.

Steadman, J. R., Coyne, D.P. and Cook, G.E. 1973. Reduction of severity of white

mold disease on great northern beans by wider row spacing and determinate plant growth

habit. Plant Disease Repertoire 57: 1070-1071.

Sun, P. and Yang, X.B. 2000. Light, temperature and moisture effects on apothecium

production of Sclerotinia sclerotiorum. Plant Disease 84:1287-1293.

Teo, B.K. and Morall, R.A.A. 1985a. Influence of matric potentials on carpogenic

germination of sclerotia of Sclerotinia sclerotiorum. I. Development of an inclined box

technique to observe apothecium production. Canadian Journal of Plant Pathology 7:359-

364.

http://www.gov.mb.ca/agriculture/crops/insects/guide-to-crop-protection.html

http://www.gov.mb.ca/agriculture/crops/insects/guide-to-crop-protection.html

7

Teo, B.K. and Morall, R.A.A. 1985b. Influence of matric potentials on carpogenic

germination of sclerotia of Sclerotinia sclerotiorum. II. A comparison of results obtained

with different techniques. Canadian Journal of Plant Pathology 7:365-369.

Teo, B.K., R.A.A. Morall, and P.R. Verma. 1989. Influence of soil moisture, seeding

date, and canola cultivars (Tobin and Westar) on the germination and rotting of sclerotia

of Sclerotinia sclerotiorum. Canadian Journal of Plant Pathology 11: 393-399.

Tu, J. C. 1989. Management of white mold of white beans in Ontario. Plant Dis. 73:281-

285.

Turkington, T.K. 1991. Factors influencing a petal-based forecasting system for

sclerotinia stem rot of canola. PhD Thesis. University of Saskatchewan, Saskatoon.

Twengstrom, E., Sigvald, R., Svensson, C., and Yuen, J. 1998. Forecasting Sclerotinia

stem rot in spring sown oilseed rape. Crop Protection 405-411.

Weiss, A., Hipps, L. E., Blad, B. L. and Steadman, J. R., 1980. Comparison of within

canopy microclimate and white mold disease (Sclerotinia sclerotiorum) development in

dry edible beans as influenced by canopy structure and irrigation. Agricultural

Meteorology 22: 11-21.

Williams, J. R. and D. Stelfox. 1979. Dispersal of ascospores of Sclerotinia sclerotiorum

in relation to sclerotinia stem stem rot of rapeseed. Plant Dis. Rep. 63: 395-399.

Wu, B.M. and Subbarao, K.V. 2008. Effects of soil temperature, moisture, and burial

depths on carpogenic germination of Sclerotinia sclerotiorum and S. minor.

Phytopathology 98:1144-1152 (Canola Council of Canada, 2011).

8

2. CANOPY DENSITY, MICROCLIMATE AND WEATHER

IMPACTS ON SCLEROTINIA STEM ROT DISEASE IN CANOLA

2.1 Abstract

Due to the lack of commercially available resistant cultivars, management of sclerotinia

stem rot (SSR) disease by fungicide application and through cultural practice is essential.

Fungicide application must be properly timed through disease forecasting which can be

accomplished through the understanding of weather impacts on SSR. More importantly,

microclimate more closely affects S. sclerotiorum development and can be modified

through canopy density alterations. Above and below canopy conditions were evaluated

for their impact on SSR from ascospore release and dispersal during flowering to disease

incidence at harvest. Three seeding rates were chosen to create variation in canopy

density and microclimate in a randomized complete block design in two Manitoba

locations (Winnipeg and Carman) during 2011 and 2012. Although cooler, moist

microclimates were experienced under denser canopies, ascospore release was not

affected. Percentage of infection was higher among denser canopies only in 1 of the 4

site-years. Ascospore concentrations were elevated almost simultaneously at both sites in

both years indicating that the regional weather conditions play an important role

considering the similar climates experienced throughout Manitoba. Peaks in daily mean

ascospore concentrations occurred after a precipitation event following conditions of

prolonged elevated relative humidity. Reduced temperatures and increased relative

humidity were responsible for the increased mean ascospore concentrations over the

sampling period and percentages of infection in Carman compared to Winnipeg. Results

9

of this study indicate that the environment plays a role in disease development, however

slight canopy density modifications are not sufficient to create favorable or unfavorable

microclimates for SSR.

10

2.2. Introduction

In western Canada, sclerotinia stem rot (SSR) is one of the most serious diseases

of canola, caused by the fungus Sclerotinia sclerotiorum (Lib.) de Bary (Manitoba

Agriculture, 2013). Considerable variation in disease incidence is evident among spring-

sown canola crops, especially in this region (Turkington and Morall, 1993) ranging from

5 to 100% (Manitoba Agriculture, 2014). Premature ripening, wilting and lodging occur

as a result of this pathogen in canola crops (Manitoba Agriculture, 2014). Due to severe

economic losses to canola crops exposed to SSR, management of the disease is

increasingly important. Primary control of SSR is through the use of fungicides, although

this method can be quite costly, especially where risks are low. It is suggested that

fungicides are to be applied once in a single event during full flowering at growth stage

65 (Lancashire et al., 1991;Twengstrom et al, 1998), however it is not always beneficial

to apply fungicides at this stage due to the sporadic nature of the pathogen (Jurke and

Fernando, 2008). To improve risk evaluation in order to better time fungicide applications

and in order to decide when fungicide application is cost effective, several researchers

have attempted to develop, evaluate and improve predictive models (Turkington et al.,

1988; Turkington et al., 1990, Turkington et al., 1991, Turkington and Morall, 1993;

Twengstrom et al., 1998; Koch et al., 2007; Bom and Boland, 2000). Better

understanding of the influence of weather factors is required to increase fungicide

application efficiency. Cultural control methods are also available as alternatives to the

use of fungicides, however their ability to reduce or control disease remains debatable.

11

Forecasting models for SSR in canola by use of petal infestations, crop density

and weather have been used in Canada (Turkington et al., 1991; Turkington and Morall,

1993). Factors such as disease incidence in the last host crop, sclerotia germination, petal

infestation, crop height, precipitation, soil moisture, leaf wetness weather forecast and

regional risk are all correlated to disease incidence (Twengstrom et al., 1998; Bom and

Boland, 2000; Clarkson et al., 2003). Gugel and Morall (1986) found a significantly

positive relationship between sclerotia germination and disease incidence in western

Canada. When weather is similar across various treatments, petal infestation is the factor

accounting for the majority of variation in disease incidence (Bom and Boland, 2000).

Petal infestations during early bloom stages have a long period for establishment on the

main stem and therefore relate to disease incidence more closely (Gugel and Moral,

1986).Weather variables that were positively correlated with high disease incidence

(>20%) were soil moisture, crop height, rainfall and presence of apothecia on the soil

surface (Bom and Boland, 2000). Precipitation occurring at the end of flowering also

increases disease incidence (Bom and Boland, 2000). Sclerotia germination has also been

modeled based on temperature to predict disease on lettuce, however sclerotia

germination was the only phase of the disease life cycle examined and therefore does not

accurately predict disease incidence on its own (Clarkson et al., 2003). In several crops,

including beans, leaf wetness and prolonged high relative humidity is required for

infection and disease development (Torés and Moreno, 1991; Abawi and Grogan, 1979).

The highest values for disease incidence in canola tend to occur during years with

extremely wet springs and early summers (Koch et al., 2007). Lower July and August

temperatures have also been linked to increased disease prevalence as sclerotinia thrives

12

in cool climates (Workneh and Xuang, 2000). Most rapid SSR development occurred at

16°C to 22°C on winter oilseed rape stems (Koch et al., 2007). Prevailing dry weather

throughout the season can be responsible for low sclerotinia incidences due to the

dependency of the disease on moisture (Morall and Dueck, 1982).

Under warm, dry conditions, misting may increase SSR disease incidence and

severity. Reduced temperature (approximately 15°C) and increased soil moisture and leaf

wetness in irrigated plots (Weiss et al., 1980) favour sclerotia germination (Teo and

Morall, 1985; Boland and Hall, 1987; Teo at al., 1989; Matheroon and Porchas, 2005;

Mila and Yang, 2008; Wu and Subbarao, 2008) and enhance development of white mold

disease in dry edible beans in semi-arid regions (Blad et al., 1978). Leaf wetness is

required for colonization, germination and infection of ascospores on nutrient containing

petal tissues for development of stem rot disease in canola (Bardin and Huang, 2001;

Huber and Gillespie, 1992). At high temperatures, reduction in spore survival on plant

tissue is possible and can reduce infection as a result (Clarkson et al. 2003). As a result of

misting, canopy structures are modified to increase crop height and leaf area index

creating denser canopies and favorable microclimatic conditions for disease development

(Blad et al., 1978).

To date, few studies have been completed on the effects of plant density and

misting on SSR in canola crops. Canopy density alterations have been proven to relate to

sclerotinia stem rot incidence in other crops including beans (Blad et al., 1978; Weiss et

al., 1980), rapeseed (Nordin et al., 1992) and several other crops (Krupinsky et al., 2002).

Proven reduction in disease incidence as a result of decreasing canopy densities was

13

demonstrated by both reducing seeding rates (Jurke and Fernando, 2008) and increasing

row spacing (Steadman et al., 1973). Lodging is increased under dense canopies

favouring within canopy moisture and contact between plants (Jurke and Fernando,

2008). Variety selection is also important in the modification of crop density and height

which are both proven to relate to disease incidence (Turkington and Morall, 1993).

Canopy density has also been included as a predictive factor in several disease forecasting

models as a contributor to disease development (Twengstrom et al., 1998; Bom and

Boland, 2000; Government of Saskatchewan, 2009; Government of Alberta, 2010). In

contrast, plant density and disease incidence were not correlated in a study conducted by

Nordin et al. (1992) in Sweden and by Koch et al. (2007) in Germany. In Koch et al.

(2007) , the number of oil seed rape plants seeded had no significant effect on disease due

to the capability for plants to fill the void space where seeding rates were decreased.

Additional study is required on the effects of misting and modified canopy densities on

disease incidence.

Release of airborne ascospores from apothecia is a critical stage in the disease

cycle. It has been debated by Harthill (1980) and Clarkson et al. (2003) whether

ascospore release occurred mainly during the day or at night. Light was required in

Harthill‟s (1980) study and spore release occurred in both light and dark conditions in

Clarkson et al.‟s (2003) study. Hourly spore release observations conclude that ascospore

dispersal occurs in a single event lasting between four to six hours daily (Qandah and

Mendoza, 2011). During wet years larger ascospore releases occurred during the day,

whereas in dry years smaller releases occurred at night (Qandah and Mendoza, 2011).

Similar to previous findings (McCartney and Lacey, 1991), ascospore release was

14

preceded by an increase in temperature (above 20°C) and drop in relative humidity

(below 80%) following periods of elevated relative humidity during wet years (Qandah

and Mendoza, 2011). Ascospore release and dispersal is largely influenced by

precipitation as shown by Qandah and Mendoza (2012), where shallower spore dispersal

gradients were generated as ascospores are capable of travelling further distances in wet

years. No spore production occurred during a dry year indicating that moisture is a major

requirement for spore release (Qandah and Mendoza, 2012). Further, mean percentage of

petal infestations determined through petal samples obtained from canola crops showed

higher petal infestations mid-day compared to the morning (Turkington et al, 1991),

which may be explained by elevated ascospore release occurring in the afternoon

(Harthill, 1980; Qandah and Mendoza, 2008). Airborne ascospore concentrations are

closely associated with the number of apothecia produced (McCartney and Lacey, 1991).

Below canopy ascospore concentrations were also positively associated with disease

incidence (Qandah and Mendoza, 2012). Above canopy ascospore concentrations have

not yet been evaluated against disease incidence and severity, nor has the influence of

microclimate on ascospore release been studied.

The objectives of this study were to determine the influence of weather on disease

development as well as to examine the impact of modified canopy density on

microclimate, ascospore release and disease incidence. This study focuses on the

modification of several microclimates using three seeding rates and three fertilizer

treatments measured to compare ascospore release and disease incidence. Ascospore

release was also compared daily during varying weather conditions during two growing

15

seasons under both misted and non-misted canola plots. Disease was then correlated to

weather and microclimatic conditions.

16

2.3 Methods

2.3.1 Study Sites

Two plot trials were established in this study during the 2011 and 2012 growing

seasons at two locations in southern Manitoba, Canada. The plots at the Carman Field

Station were situated on a well-drained clay loam soil (Manitoba Agriculture, 2013). The

plots at “The Point”, University of Manitoba, Winnipeg were on clay soil (Manitoba

Agriculture, 2014).

The history of each of the sites used in this study is important as the type of crops

grown and past disease incidence is essential in understanding the results from the study.

The Carman 2011 plot had been seeded with soybean in 2010 and sunflower in 2009,

both of which are susceptible to Sclerotinia sclerotiorum. Sclerotinia stem rot disease was

found throughout the field in 2010 and was therefore likely still present in 2011. The

Winnipeg 2011 plot had been seeded with canola in 2008, flax and winter wheat in 2009

and winter wheat in 2010. No sclerotia were identified. Flax and wheat are non-host crops

of Sclerotinia sclerotiorum. In 2012, the plots used in both locations had been planted to

non-host crops in 2011, flax in Carman and wheat in Winnipeg.

2.3.2 Field Preparation

2.3.2.1 2011 Growing season

In 2011, seeding in Winnipeg took place on May 26th. The portion of the field

being used was approximately 34 metres wide by 120 meters long. Prior to seeding, the

field was sprayed with glyphosate to eliminate weeds and the seeds were treated with

Helix XTra ® treatment to control flea beetles (Phyllotreta spp.). The field received no

17

tillage. Fungicides were not applied to ensure the presence of sclerotinia. The south

portion of the field (34m wide by 60m long) was seeded with Brassica napus canola

(Westar). The canola was divided into 18 equal-sized plots that were 10m by 10m, with

2m gaps between each plot running from west to east (Figure A.4). The plots contained 3

seeding density treatments in 3 replicates in a randomized-complete block design and

used two precipitation environments (natural rainfall only and wet continuously with a

misting system). Seed and fertilizer rates are shown in Table 2.1. Fertilizer rates applied

to the soil were determined based on recommendations provided by Agvise Laboratories

Inc. Soil samples were taken from 0 to 6 inches and 6 to 24 inches on May 6th

, 2011 and

sent to Agvise for analysis of soil nutrient status.

Due to technical difficulties, the south 2m of plots 7 (low seeding) and 8 (medium

seeding) in Winnipeg were incorrectly seeded with a high seeding rate in the plot

allocated as “low seeding” and no seed in the plot allocated as “medium seeding”. Plot 8,

allocated as medium seeding was seeded by hand on June 10th

, 2011. These modifications

should not have impacted the overall analysis.

In 2011, the Carman plot was seeded June 8th

. The site was approximately 54 by

60 metres in size. Prior to seeding, the field was conventionally tilled. No herbicides were

applied to the field prior to seeding and the seeds were treated using Helix XTra

treatment. No fungicides were applied to ensure the presence of disease. A 30m wide by

70m long area was seeded to Westar, which was similarly divided into 18 equal plots (10

m by 10m). Two meter gaps separated each plot. Fertilizer rates applied were similar to

18

the rates applied in Winnipeg and based on recommendations provided by Agvise

Laboratories Inc. (Table 2.1).

No seed was applied to a 2 m wide strip in plot 1 (high seeding) and a very high

seeding rate was applied to a 2m strip in plot 6 (medium) in Carman because the cone on

the seeder was incorrectly tripped. These changes should not have impacted the overall

analysis.

Table 2.1 Seeding and fertilizer treatment rates for Winnipeg and Carman field sites in

2011 and 2012.

Canopy Density

Treatment

Seeding

Rate

(lb/ac)

Fertilizer Type Fertilizer

Rate (kg/ha)

Canola Low 5.53 22-0-0-24 (Ammonium Sulphate) 63

11-52-0 (MAP) 25

46-0-0 (Urea) 25

Medium 11.06 22-0-0-24 (Ammonium Sulphate) 63

11-52-0 (MAP) 25

46-0-0 (Urea) 84

High 16.59 22-0-0-24 (Ammonium Sulphate) 63

11-52-0 (MAP) 25

46-0-0 (Urea) 168

19

2.3.2.2 2012 Growing Season

Fall field preparation for both sites for the 2012 growing season was completed by

November 2nd

, 2011. Carman and Winnipeg field layouts were similar to those in 2011 at

each location. Pre-emergent Edge was applied to the soil at both the Carman and

Winnipeg canola portions of the fields on October 19th and 11

th respectively for weed

control in 2012. A harrow was then used to trap granules at the soil surface directly after

the application of Edge. One week later, the fields were harrowed again at a 90 degree

angle to further incorporate the herbicide.

During the spring of 2012, roundup was applied to the field in Winnipeg for weed

control and Carman received tillage during the spring prior to the growing season. Similar

to 2011, seeds were treated with Helix seed treatment and seeding and fertilizer rates

remained the same (Table 2.1). Winnipeg and Carman were seeded on June 6th and May

23rd

respectively. Equinox EC with merge surfactant was applied to the canola in Carman

on June 22nd

to eliminate grassy weeds.

Due to technical difficulties operating the misting system, no misting occurred in

2012 at either field location. Thus, there were two identical sets of plots at each location,

each with 3 replications of high, medium and low crop densities following a randomized-

complete block design under natural environmental conditions (Figure 2.1).

20

Figure 2.1 Canola field set up for Winnipeg and Carman in 2011 (left) and 2012 (right).

High, medium and low seeding and fertilizer rates are represented by dark grey,

light grey and white shades within canola plots.

21

2.3.3 Inoculation

Both the Carman and Winnipeg sites were inoculated with sclerotia after seeding

to ensure the presence of the pathogen. Since sclerotia require conditioning throughout

the winter, the inoculum was placed outside in January, 2011 inside nylon mesh bags and

then covered by snow for the duration of winter. Upon snowmelt, the mesh bags were

removed from the soil and placed in a refrigerator to avoid sclerotial germination.

On June 6th

2011, sclerotia were taken out of the refrigerator and weighed. The

experiment at The Point in Winnipeg was inoculated with 60 grams of sclerotia placed on

each of the 18 canola plots. The sclerotia were spread by hand by tossing the inoculum

into each of the plots. There was no known amount of sclerotia present within this field

from previous years. Inoculation of sclerotia in Carman occurred on June 15th, 2011 with

60 grams of sclerotia placed within each of the 18 canola plots. There were sclerotia

present in this field from the 2010 growing season, however the amount of sclerotia from

the previous year was unknown.

For the 2012 growing season, fall field preparation in 2011 included the

inoculation of sclerotia onto each of the Winnipeg (November 2nd

, 2011) and Carman

(October 26th, 2011) field sites. Equal amounts of sclerotia were applied by hand to both

fields in the fall to allow conditioning to occur throughout the winter. In both fields, 66g

of sclerotia was applied to each of the 18 canola plots. During the spring of 2012, an

additional 40g of sclerotia was applied to each of the 18 canola plots in Winnipeg and

Carman on June 5th and June 14

th respectively.

22

2.3.4 Microclimate Monitoring

Microclimate was monitored throughout each individual plot during the two

growing seasons using individual HOBO microclimate stations in each plot (Figures A.3,

A.4 and A.5). Microclimate measures included air temperature, relative humidity, leaf

wetness and surface soil temperature and moisture. One microclimate station was placed

in each of the plots situated within the canola fields (Figure 2.1). In 2011, the

microclimate stations were placed in the fields in Winnipeg and Carman on June 6th

and

June 17th

respectively, once the crops had emerged and were at the seedling stage. In

2012, stations were installed in the Winnipeg and Carman fields on June 26th

and June

13th respectively. Soil moisture and temperature probes were placed in the soil vertically

using a trenching shovel to dig the holes to 8 cm and the surrounding area was replaced

with soil. The radiation shield, containing the relative humidity and air temperature probe,

was placed facing northwards at approximately 10 cm above the soil surface to account

for microclimate conditions near the surface at both Carman and Winnipeg locations.

Microclimate stations were placed in the center of each plot in 2011 and a quarter way

into the plot in 2012 (Figure A.4).

Leaf wetness sensors and relative humidity and temperature sensors within the

solar radiation shield were adjusted throughout both growing seasons. Leaf wetness,

temperature and relative humidity sensors were placed at 12 cm in Winnipeg and Carman

on June 28th

and July 17th respectively in 2011 and on June 26

th and June 13

th respectively

in 2012. In 2011, leaf wetness, temperature and relative humidity sensors were raised to

23

24 cm on July 7th

(Winnipeg) and July 21st (Carman). In 2012, sensors were placed at 24

cm on July 4th (Winnipeg) and June 21st (Carman).

The presence of weeds within each plot affected canola growth and may also have

impacted microclimate. In 2011, at Winnipeg, weeds were present in strips within the

plots running through the microclimate stations locations in several plots. Plots were

characterized as having high, medium and low weed presence. Plots with heavy weed

presence included plots 2, 5, 6, 8, 14 and 18. Some weeds were found in plots 1, 7, 9, 11,

12, 16 and 17 while little to no weeds were found in plots 3, 4 and 15. During the

flowering period, weeds were less apparent. In Carman 2011, weeds were heavily present

in plots 11, 13 and 15 while some weeds were present in plots 1 and 10. Little to no

weeds were observed among the remainder of the plots in Carman. Due to the presence of

heavy weed infestations in Carman in some plots where microclimate stations were

situated, stations were moved on July 12th, 2011 at 10:00 am to better represent

microclimatic conditions in canola among the plots. In 2012, weeds in Carman were

sufficiently controlled to avoid any impacts to canola growth and microclimate

monitoring. In Winnipeg, the majority of weeds were present in plots 13, 14, 16, 17and

18 and were characterized as having a medium weed presence.

Surface soil volumetric water content (m3/m

3) was measured using soil moisture

smart sensors (S-SMC-M005) that work with HOBO data loggers to determine the

dielectric constant in the soil.

24

In order to calibrate the soil moisture smart sensors, soil samples were taken

during four sampling periods at each site throughout each growing season. In 2011,

during a soil sampling period, several gravimetric and volumetric soil samples were taken

throughout the field. Three samples were taken in every plot next to each soil moisture

smart sensor. A volumetric soil surface sample was taken by scooping soil into a labelled

aluminum tin and covering the sample with a lid to avoid evaporation. A gravimetric soil

surface sample was taken by scooping a sample of surface soil into a labelled plastic bag

using a small shovel. The number of volumetric and gravimetric samples taken per plot

varied due to the amount of aluminum tins available, although at least one volumetric

sample was taken per plot. Samples were then placed in a cooler and brought to the lab

for further analysis. Soil samples were weighed and dried in an oven at 105 degrees

Celsius for 24 hours, then weighed again to get a measure for gravimetric water content.

Volumetric water content was also determined for some of the samples by incorporating

the known volume of the aluminum tin into the calculation. In 2012, only volumetric

samples were taken using aluminum cores. Cores containing the samples were placed in a

larger tin with a lid and brought back to the lab in a cooler. Volumetric soil moisture

content was also calculated using the weighing and drying method as described above.

The volume of the core was incorporated in the calculation.

Calculations for gravimetric and volumetric soil moisture content are as follows:

Gravimetric moisture content

GM = ( WS(g) – DS(g) ) / DS(g) (1)

25

Where GM is gravimetric moisture content, WS is the wet soil before drying in grams and

DS is the dry soil after drying in grams. All three gravimetric values per plot were

averaged to get average gravimetric soil moisture content values per plot.

Bulk Density (g/cm3)

BD(g/cm3) =DS(g) / V(cm

3) (2)

Where BD is bulk density V is the volume of the aluminum tin or core containing the soil.

An average bulk density (g/cm3) was obtained for the entire field.

Volumetric Moisture Content

VM(g/cm3)= BD(g/cm

3) x GM (3)

1 g/cm3 = 1m

3/m

3

2.3.5 Weather Station Monitoring

Standard weather data was monitored at both locations throughout the growing

season. Weather data was recorded at The Point in Winnipeg from June 2nd

, 2011 through

August 8th, 2011, and from June 1

st, 2011 through August 26

th, 2011 in Carman at the Ian

N. Morrison Field Station. A Campbell Scientific Inc. weather station was set up on the

southwest corner of field 8 located in Winnipeg on June 1st, 2011. Air temperature (˚C),

26

relative humidity (%), wind speed (km/h), solar radiation (W/m2 and MJ/m

2) and

precipitation (mm) were measured through this station. The air temperature and relative

humidity probe was enclosed in a radiation shield and was placed at a height of 1.5m

above the soil surface. The anemometer was placed at a height of 3m and pyranometer at

2m above the soil surface. A tipping bucket rain gauge was placed alongside the weather

station at a height of 30cm above the surface. Each of the instruments was hooked up to a

data logger (10X) which averaged data hourly and daily. A watchdog station was also

placed alongside the weather station as a backup and to obtain additional data for wind

direction (degrees) through the use of a wind vane. In Carman, similar weather data was

obtained online from the Carman Field Station, less than 2 km from the field through a

weather station that was previously set up by Manitoba Agriculture, Food and Rural

Development (MAFRD). A tipping bucket rain gauge was also placed alongside the field

at 30cm above the soil surface in Carman to serve as backup for precipitation data.

Due to technical issues with the Watchdog Station at The Point in Winnipeg,

Watchdog data is only available from June 2, 2011 through June 29th

, 2011 and July 27th,

2011 through August 8th, 2011.

2.3.6 Ascospore Dispersal

2.3.6.1 Rotorod Spore Sampler

The release and dispersal of spores by apothecia was monitored through the use of

rotorods (Multidata LLC) (Figure A.6). The rotorod spore sampler is a device containing

a sampling head which holds 2 polystyrene collector rods. The sampler was operated on a

timer that intermittently spun the rods in a clockwise direction at a speed of 2400 RPM

27

capturing airborne spores. The rods spin for 1 minute and rest for 9 minutes, therefore

collecting fungal spores 10 percent of the time. The surface of the rod facing the direction

of the wind while spinning was marked as the leading edge and pre-greased with a silicon

solution to provide a “sticky” surface for the collection of spores. Rods were greased in

the laboratory by holding one end of the rod (the end being placed into the spinning head)

and applying the silicon grease to the one side by hand ensuring the silicon was spread as

evenly and thinly as possible. Each rotorod was interconnected by 10 and 12 gauge

electrical wiring and hooked up to a timer, which is connected to a 12 volt battery. The

battery was also connected to a solar panel which provided additional power and

recharged the battery.

In each field, one spore sampler was placed in each of the 18 canola plots to

monitor spore dispersal variation among the various crop densities. Rotorods placed

within canola plots were elevated directly above the crops. All rotorod samplers placed in

Winnipeg contained retracting heads that exposed the spore collecting rods only when the

heads were spinning. Rotorods placed in Carman contained fixed heads in which the

spore collecting rods are always facing downwards. Rods were replaced daily and placed

in centrifuge tubes for further analysis. In 2011, rotorods were set up in Winnipeg on June

20th and in Carman on July 5

th. In 2012, rotorods were set up and monitored beginning on

June 27th

in Carman and July 7th

in Winnipeg.

2.3.6.2 Collector Rod Sample Assessment

The rods removed from the field were analyzed under a microscope at 400x

magnification and fungal ascospores of S. sclerotiorum were counted. Identification was

28

based on ascospore morphology. Ascospores are single-celled, hyaline in appearance, bi-

nucleate and are ellipsoid in shape and 4-6 x 9-14 µm in size (Kohn, 1979). Spores were

not counted if they could not be identified. Since the collector rods recovered so many

spores and other particles, analyzing an entire rod was impractical therefore an

abbreviated analysis was done. A blank slide was first prepared by permanently marking

a solid line at each 1.375 mm, creating sixteen 2.09 mm2 sampling areas. Sclerotinia

sclerotiorum ascospores were then counted within the circular lens view area once in each

sampling area. Only ten of sixteen sampling areas were used because the first six were

generally those with the black mark that was placed and held within the sampling head of

the rotorods. The total number of ascospores per rod was then calculated based on the

counts obtained in each lens view by the following calculations:

Circular lens view area of microscope formula:

A = л (r)2 (4)

A1 = л (0.275 mm)2 = 0.238 mm

2 (Olympus BX51)

A2 = л (0.24 mm)2 = 0.181 mm

2 (BH-2)

A3 = л(0.24 mm)2 = 0.181 mm

2 (Hund Wetzlar Typ H 600/12)

Where “A1, A2 and A3” are the lens view areas of the circle for microscope 1 (Olympus

BX51), microscope 2 (BH-2) and microscope 3 (), “л” is pi (3.14159265) and “r” is the

radius of the lens view.

Total area for all 10 spore counts:

29

A1(10) = 0.238 mm2 (10) = 2.38 mm

2 (Olympus BX51) (5)

A2(10) = 0.181 mm2 (10) = 1.81 mm

2 (BH-2)

A3(10) = 0.181 mm2 (10) = 1.81 mm

2 (Hund Wetzlar Typ H 600/12)

Where “A(10)” is the total area for all 10 spore counts.

Multiplication factor for entire rod for each microscope:

M1 = 22 mm2 / 2.38 mm

2 = 9.24 (Olympus BX51) (6)

M2 = 22 mm2 / 1.81 mm

2 = 12.15 (BH-2)

M3 = 22 mm2 / 1.81 mm

2 = 12.15 (Hund Wetzlar Typ H 600/12)

Where M1, M2 and M3 are multiplication factors used to calculate total spore counts. The

spore counts for each area were then multiplied by the multiplication factors (M1, M2 and

M3) to get the total number of spores for each rod.

TS = S(10) x M1 (Olympus BX51) (7)

TS = S(10) x M2 (BH-2)

TS = S(10) x M3 (Hund Wetzlar Typ H 600/12)

Where TS is the total spore count for a rod, S(10) is the spore count over 10 lens view

areas.

The total spore count was then divided by the volume of air sampled (3.12 m3).

Occasionally samples were taken slightly before or after one day therefore the number of

30

minutes during the sampling period was divided by the total minutes in a day (1440) and

multiplied with the volume of air sampled. The counts for each rod were then divided by

the resulting volume of air sampled.

The values obtained for each of the two rods were then averaged to get a mean

value of ascospores per metre cubed per rotorod per day:

Ascospores/m3 = (TS(rod 1) + TS(rod 2)) / 2 (8)

Occasionally rods could not be counted or contained no spores for several reasons.

Rods that could not be counted properly had lots of dirt on them, large sections of the rod

were covered by a bug or large sections of the rod contained no spores. Rods containing

either no spores or dirt on them were generally those that had been placed in the rotorod

backwards and also could not be counted. Some rods were also missing meaning that they

may have been dropped in the field and not found. Where only one of the two rods from a

rotorod was counted, the value from that rod alone was counted as the mean daily

ascospore concentration. Where neither of the two rods could be counted, gaps in the data

resulted.

2.3.7 Petal Sampling

Canola petal samples were taken twice throughout the flowering period, at early

and late bloom stages, at both locations to determine whether ascospores were present on

the petals. In 2011, samples were taken at 30 and 70% bloom, in Winnipeg on July 6th

and 14th

and in Carman July 18th

and 25th

. Sixteen flowers were sampled at random in

31

each plot, from plots 1 through 18 at both locations. The lowermost flowers were taken in

eight of the sixteen flower samples in each plot, while the uppermost flowers were taken

for the remaining eight samples. In 2012, nine random samples were taken per plot at

each site during each sampling period; at 15%, 30% and 70% bloom. Samples were taken

on July 5th, July 9

th and July 18

th in Carman and on July 13

th, July 19

th and July 24

th in

Winnipeg. Each flower was cut from the stems using scissors, and placed into a small

labelled freezer bag within a cooler containing ice, then brought to the lab to be plated.

In 2011, a Rose Bengal Agar was used as the medium for plating petals. One petal

from each flower was plated on Rose Bengal Agar (Bom and Boland, 2000) immediately

after sampling. As per Bom and Boland (2000), Rose Bengal Agar was prepared by first

mixing 20 grams per litre (g/L) potato dextrose agar (Difco) with 30 parts per million

(ppm) Rose Bengal. The mixture was then autoclaved for 15-20 minutes. 20 parts per

million (ppm) of streptomycin sulphate was added after cooling. In 2012, a semi-selective

media containing bromophenol blue was used for the petals to improve the ability to

identify ascospores of S. sclerotiorum (Steadman et al., 1994). At a pH of 4.7 or lower,

bromophenol blue changes from blue to yellow; a color change caused by the presence of

S. sclerotiorum due to the production of oxalic acid. A solution of 39g/L potato dextrose

agar (Difco), 25 mg/L of 75% pentachlorobenzene (PCNB),150 mg/L of penicillin G,

150 mg/L of streptomycin sulfate and 50 mg/L of bromophenol blue was made. Potato

dextrose agar was autoclaved for 15-20 minutes before the remaining sterilized

ingredients were added. The prepared solutions were then poured into plates and left to

cool. Within a fume hood, four individual petals were cultured on each labelled plate

using forceps to pull the petals from each flower. Petals were then placed on the medium

32

while ensuring that the entire surface of each petal was touching the agar medium. Prior

to plating each petal, forceps were dipped in an alcohol solution and flamed to remove

any contamination. Plates were left at room temperature for 14 days. Observations were

made after 14 days and colonies of S. sclerotiorum were identified.

For petals plated in 2011 on Rose Bengal Agar, petals containing S. sclerotiorum

ascospores were described as having a colony of white loosely interwoven mycelium

filaments, large black fruiting bodies (sclerotia) produced above the mycelium and

adjacent agar medium turning from pink to clear. Petals that did not contain ascospores

were frequently not diseased and only the petal on the rose Bengal agar was observed.

Sometimes yellow, green or white fluffy mycelium was present which represented other

fungi and were not characteristic of Sclerotinia ascospores. Several plates that did not

contain ascospores also contained small fruiting bodies as well as adjacent agar medium

turning from pink to orange. White-grey or colonies present on the agar plates did not

originate from the petals, and were contaminants likely introduced while plating the

petals.

Petals plated on the semi-selective media in 2012 containing S. sclerotiorum

ascospores were identified by observing a color change in the medium from blue to

yellow. Since other fungal pathogens also create this color change including Penicillium

and Aspergillis, sclerotia must also be found on the plates in order to positively identify S.

sclerotiorum.

33

2.3.8 Misting

During the 2011 growing season, half of the canola plots in each field were misted

to ensure continual moisture for disease development and to provide a range of

microclimatic conditions. The remaining plots were not misted to represent standard

weather conditions. Misting occurred in Winnipeg plots 10 through 18, 1 through 9 in

Carman. Misting in 2011 began at the 20% bloom stage on July 5th and July 15

th in

Winnipeg and Carman respectively. Ten days after the final canola petal had fallen,

misting ceased on July 31st (Winnipeg) and August 10

th (Carman). Misting began daily at

4 pm and was left on until the 1,200 gallons (4,542 L) emptied. Approximately 26,250

gallons (99,367 L) of water was irrigated onto the misted field portions in total.

The misting system at each site consisted of a tank capable of holding 1,250

gallons (4,732 L), a pump and a generator operated with fuel to provide energy to the

pump. The pump contained a timer which allowed for misting to occur for 5 minutes out

of every hour. The system was set up so that piping ran from the pump to the misted field

portions. Each misting head was secured to a pole, 4.5 feet (1.37 metres) high, which was

placed in the soil where misting was required. A single misting head was capable of

misting up to a radius of 2.5 meters, covering an area of approximately 19.6 square

meters total. Four misting heads were placed at equal distance in each plot, each being 5

meters apart.

Neither the Winnipeg nor Carman plots were misted in 2012 due to technical

issues and availability of generators and pumping systems. This created two sets of

completely randomized block designs in each of the Winnipeg and Carman fields in 2012.

34

2.3.9 Crop Development

Crop development of canola was observed throughout each growing season. The

development stage and the date at which each stage was observed in the field are listed in

Table 2.2 and illustrated in Figures 2.2 and 2.3. Development stages are approximated to

when the fields were visited. There was slight variation among growth stages within

different canopy densities in each field. Growth stages for canola were based on those

described by the Canola Council of Canada (2011).

Table 2.2 Growth stages of Canola and date at which these stages were initially observed at Carman and in Winnipeg.

Winnipeg Carman

Year Development Stage Day Day

2011z Seedling Cotyledon June 8th June 15th

Rosette Stage (with Cotyledons and Second True Leaves) June 13th June 20th

Rosette Stage (4th Leaf) June 15th June 29th

Budding stage June 29th July 9th

Flowering stage July 4th July 14th

End of Flowering stage July 21st August 6th

2012y Seedling Cotyledon June 10th May 31st

Rosette Stage (with Cotyledons and Second True Leaves) June 22nd June 9th

Rosette Stage (4th Leaf) June 26th June 16th

Budding stage July 6th June 26th

Flowering stage July 10th July 1st

End of Flowering stage Aug 1st July 22nd

z Winnipeg and Carman sites were seeded on May 26th and June 8th respectively.

y Carman and Winnipeg sites were seeded on May 23rd

and June 6th respectively.

Figure 2.2 Diagram of canola growth stages in Winnipeg and Carman in 2011.

35

Figure 2.3 Diagram of canola growth stages in Winnipeg and Carman sites in 2012.

36

2.4.0 Canopy Density

Canopy thickness or density was determined using an LAI 2200 meter and by

counting plants within 1m2 quadrats at both the Winnipeg and Carman locations. Average

canopy densities were also compared between misted and non-misted field portions in

2011.

The LAI 2200 Plant Canopy Analyzer was used throughout the growing season to

determine leaf area index (LAI). Leaf area index (LAI) is a measure of the leaf surface

area (1 side) per unit area of the ground with values typically ranging from 0 to 6 for bare

ground to very dense canopy, respectively. Each of the 18 canola plots at both locations

were monitored four and five times in Winnipeg and Carman respectively in 2011. In

Winnipeg, measurements were taken on June 30th

, July 5th

, July 12th

and July 21st 2011.

Measurements were taken on July 8th

, July 11th, July 19

th, July 27

th and August 3

rd, 2011

in Carman. In 2012, four measurements were taken in Winnipeg and Carman. Winnipeg

measurements were taken on July 9th

, July 13th

, July 19th and July 26

th and Carman

measurements were taken on June 25th, June 29

th, July 10

th and July 17

th.Within each

canola plot, one LAI measurement was taken above the canopy, and four LAI

measurements are taken below the canopy. In each plot one measurement was taken

approximately 2.5m inside from each of the plot corners. An average LAI was then

calculated based on the four below canopy measurements and one above canopy

measurement. Measurements were generally taken before 11:30 to ensure the sensor was

shaded by the sampler while taking individual measurements. Each measurement was

taken at the same angle, with the sampler blocking the sun from the sensor and no view

caps were used. The plant canopy analyzer sensor was placed horizontally on the surface

37

below the canopy for each below canopy reading, while ensuring that there were no

leaves directly above the sensor.

Four 1m2 quadrats were placed in each plot at both locations prior to swathing in

order to compare canopy densities. The total number of plants per metre square was

counted in each of the quadrats. The four quadrats in each plot were then averaged to get

a value of plants per meter squared representative of each plot. Plants were counted on

August 5th, 2011 and August 7

th, 2011 in Winnipeg and Carman respectively and on

August 13th, 2012 and August 7

th, 2012 in Winnipeg and Carman respectively. An

additional plant count was completed on August 22nd

, 2012 where two quadrats were

placed in each plot.

2.4.1 Disease Incidence

Disease incidence was evaluated in Carman and Winnipeg prior to swathing by

determining the percentage of S. sclerotiorum infected canola plants to the total number

of canola plants. In 2011, disease assessments were done on August 5th and August 22

nd

in Winnipeg and Carman while 2012 disease assessments were done on August 13th and

August 7th, respectively. Four one meter square quadrats were placed in each plot at

random locations and disease counts were made. The total number of canola plants and

number of plants infected by S. sclerotiorum were counted in each quadrat. The mean of

all four quadrats in each plot was determined to get a representative percentage value for

disease incidence (Jurke and Fernando, 2008). An additional abbreviated disease

assessment was done on August 22nd

, using two quadrats in each plot.

38

Diseased canola plants were identified as having bleached stems with pale grey

and white lesions on stems, branches and pods. Canola plants that contained the above

symptoms were also counted if they were lodging. Infected plants were also checked to

determine if they were brittle and shredded (Manitoba Agriculture, 2013). Blackleg was

another canola disease present at both the Winnipeg and Carman sites. Plants with

blackleg were buff coloured, contained spots and small black fruiting bodies in the stems

(Manitoba Agriculture, 2014). Fusarium wilt causes canola to have purple, grey or brown

streaks running throughout the plant. Generally only 1 side of the plant or several

branches will show symptoms of Fusarium Wilt. Only plants with symptoms for

Sclerototinia stem rot disease were counted.

2.4.2 Data Analysis

Average, total, minimum and maximum values for canopy density, weather

(precipitation, temperature, relative humidity and solar radiation), microclimate

(temperature, relative humidity and leaf wetness) and disease development parameters

were tabulated and summarized for comparison among site-years. Comparisons between

Winnipeg and Carman for parameters including ascospore concentrations, precipitation,

relative humidity, temperature, incoming solar radiation and wind speed were made based

on timely trends observed in figures. Precipitation and ascospore concentrations were

accumulated during the flowering period and compared. The effects of precipitation and

misting were analyzed against ascospore concentration and disease development. Other

weather and microclimate factors including temperature (above and below canopy),

relative humidity (above and below canopy), solar radiation, wind speed, soil temperature

and leaf wetness were also compared with ascospore concentrations and disease

39

incidence. Ascospore concentrations that fell between certain thresholds were averaged

and graphed to demonstrate weather impacts on atmospheric spore concentrations.

A factorial analysis of variance where all cell means are compared simultaneously

through a 2 x 2 x 3 design was used to examine the effects of year, location and seeding

rate on the dependent variables. Dependent variables include canopy density (LAI and

plant counts), disease development (ascospore concentration and percentage of infection)

and microclimatic measurements. Dependent variables were also compared in a factorial

analysis with location, misting and seeding rate as independent variables using data

obtained from 2011. Statistical Analysis System 9.2 (SAS Institute Inc. Cary, NC), was

used for the analysis of variance (PROC MIXED) for all 8 dependent variables at p =

0.05. Custom comparisons were made using contrasts. Tukey‟s HSD (honestly

significantly different) test using a pair-wise comparison of differences in least squares

means was used for comparison of treatment means for equal sample sizes. A Tukey-

Kramer test was used for unequal sample sizes where data were missing and individual

plots were removed from the analysis.

40

2.4 Results

2.4.1 Summary of Results

The growing season, sampling period, bloom stage and ripening period dates for

each site year are outlined in Table 2.3. Monthly mean temperature and total precipitation

compared to monthly means from 1981 to 2010 are shown in table 2.4. Temperatures

were above normal throughout the season in all 4 site years. Precipitation was below

normal in all four site years during all months except for June in Winnipeg 2011 and

Carman 2012. Measurements for canopy density, weather, ascospore concentrations as

well as percentage of infection are summarized in Table 2.5. Overall, highest LAI values

were in Carman 2011. Generally early bloom LAI was higher compared to late bloom

with the exception of Carman 2011. The highest mean plant counts were obtained in

Winnipeg in 2011 while higher plant counts occurred in 2011 within each plot at both

Carman and Winnipeg. Average mean infection percentages were higher in 2011 overall

compared to 2012, with the highest percentages occurring in Carman compared to

Winnipeg during both years. Average mean ascospore concentrations were higher in 2012

overall, with significantly higher values in Carman than Winnipeg in 2012. Higher

maximum mean daily ascospore concentrations were found in Carman during both years.

The late bloom stages in all site years showed higher average, minimum and maximum

mean daily ascospore concentrations than early bloom stages. During the growing season,

total precipitation showed the highest values in Carman 2012. Higher values were

reported for Winnipeg in 2011 than in 2012. The 2012 sampling period received more

rain than 2011 with higher values in Winnipeg during both years. Total precipitation

41

during late bloom was higher than in early bloom during all site years. Precipitation was

higher during 2012 towards harvest compared to 2011. Average mean daily air

temperatures were relatively consistent throughout the growing season however, in 2012

Winnipeg experienced slightly higher temperatures than Carman. During late bloom and

ripening, Winnipeg also experienced higher temperatures than Carman with minimum

and maximum values also being higher. Average values for daily mean relative humidity

were higher in Carman than Winnipeg throughout each growing season during both years.

There was no consistent difference in relative humidity between early and late bloom

stages. During the growing season, average daily mean wind speed was lowest in

Winnipeg in 2012 compared to other site-years when a maximum mean daily speed of

only 3.3 m/s was achieved. Average total daily incoming radiation was relatively similar

across all site years. Slightly higher incoming solar radiation was experienced in

Winnipeg during late bloom compared to Carman during both years.

42

Table 2.3 Dates for growing season, sampling period, ripening, early and late bloom stages during all four site years.

Growing Season Sampling Period Early Bloom Late Bloom Ripening

Carman 2011 June 8 - August 22 July 11 - August 17 July 14 - July 25 July 26 - August 6 August 6 – August 22

Winnipeg 2011 May 26 - August 5 July 4 - July 29 July 4 - July 12 July 13 - July 21 July 21 – August 5

Carman 2012 May 23 - August 7 July 8 - August 6 July 1 - July 11 July 12 - July 22 July 22 – August 7

Winnipeg 2012 June 6 - August 13 June 27 - July 24 July 10 - July 20 July 24 - August 1 August 1 – August 13

43

Table 2.4 Monthly mean temperature and total precipitation and long term monthly means in Winnipeg and Carman in 2011

and 2012.

2011 2012

Site Month Long-term meanz Monthly Percent of Mean (%) Monthly Percent of Mean (%)

Temperature (°C)

Winnipeg May 11.6 - - - -

June 17 18.2 1.2 19.1 2.1

July 19.7 22.2 2.5 24 4.3

August 18.8 - - - -

Carman May 11.6 - - 12.5 0.9

June 17.2 17.3 0.1 18.4 1.2

July 19.4 21 1.6 22.6 3.0

August 18.5 - - 19.4 0.9

Precipitation (mm)

Winnipeg May 54.1 - - - -

June 90 114.3 127 68.3 76

July 79.5 37.3 47 24 30

August 77 - - - -

Carman May 67.7 - - 31.6 47

June 96.4 81.2 84 110 114

July 78.6 45.2 58 35 45

August 74.8 - - 56.2 75

z Government of Canada. 2014. Climate.1981-2010 Climate Normals and Averages. Climate.weather.gc.ca/climate_normal/index_e.html

44

Table 2.5 Summary of measurements for canopy density (LAI, plant counts), weather parameters, ascospore concentrations and percentage

of infection during the growing season, sampling period, ripening, early bloom and late bloom for all four site years.

Year Location Period

Total

Precipitation

(mm)

Mean Daily Air

Temperature

(°C)

Growing

Degree

Days

Mean

Relative

Humidity

(%)

Mean Wind

Speed (m/s)

Total Solar

Radiation

(kW/m2)

Mean

LAI

Mean

Plant

Counts

(Plants/m2)

Mean

Ascospore

Concentrations

(ascospores/m2)

Mean

Percent

Infection

(%)

2011 Carman Growing Season 135z 20 (12,26) 1441 72 (54,92) 2.4 (1.0,5.4) 6.1 (1.0,8.5) 3.46 92 ˗ 22.5

Sampling Period 24z ˗ 767 ˗ ˗ ˗ ˗ ˗ 595 (64,2760) ˗

Early Bloom 5z 22 (15,26) 257 74 (54,92) 2.4 (1.1,3.7) 6.4 (3.5,8.1) 3.81 ˗ 398 (64,1332) ˗

Late Bloom 11z 21 (18,23) 247 73 (64,83) 2.0 (1.0,3.3) 6.3 (3.9,7.9) 3.91 ˗ 765 (203, 2760) ˗

Ripening 8 19 (15,22) ˗ 71 (59,81) ˗ ˗ ˗ ˗ ˗ ˗

2011 Winnipeg Growing Season 162z 20 (10,28) 1398 64 (40,90) 2.6 (0.6,6.0) 6.1 (1.6,8.5) 3.04 141 ˗ 5.5


Early Bloom 27z 22 (18,25) 192 61 (51,71) 2.1 (1.5,2.9) 7.1 (5.3,8.4) 3.18 ˗ 251 (35,640) ˗

Late Bloom 34z 25 (19,28) 217 62 (45,74) 2.4 (0.6,4.8) 6.8 (5.2,8.4) 2.13 ˗ 486 (55,1201) ˗

Ripening 14 22 (16,25) ˗ 61 (45,71) ˗ ˗ ˗ ˗ ˗ ˗

2012 Carman Growing Season 208z 19 (6,26) 1458 70 (46,90) 2.4 (0.8,5.1) 6.0 (0.9,9.0) 3.05 111 ˗ 15.1


Early Bloom 14z 23 (21,26 255 69 (63,77) 1.6 (0.8,2.4) 6.4 (2.6,8.7) 3.44 ˗ 669 (24,2685) ˗

Late Bloom 21z 22 (17,25) 245 75 (62,87) 2.0 (1.1,3.2) 6.0 (2.9,8.4) 3.17 ˗ 1961 (127,5195) ˗

Ripening 27 21 (16,25) ˗ 94 (91,98) ˗ ˗ ˗ ˗ ˗ ˗

2012 Winnipeg Growing Season 129z 22 (11,27) 1459 64 (46,87) 1.8 (0.6,3.3) 6.1 (1.7,8.8) 3.08 76 ˗ 3.6


Early Bloom 7z 24 (19,27) 261 64 (53,81) 2.0 (0.8,2.9) 5.7 (1.2,8.0) 3.34 ˗ 569 (32,1169) ˗

Late Bloom 10z 24 (20,26) 285 60 (53,71) 1.7 (0.8,2.8) 6.5 (4.1,7.8) 2.86 ˗ 801 (111,2850) ˗

Ripening 39 20 (16,23) ˗ 64 (52,78) ˗ ˗ ˗ ˗ ˗ ˗

zRepresents total values

Values in parenthesis represent min and max respectively

45

2.4.2 Weather Impacts on Ascospore Concentrations

2.4.2.1 General Weather Patterns

Comparisons of ascospore concentrations and weather parameters were made

between the Carman and Winnipeg plots in 2011 and 2012 (Figures 2.4 and 2.5).

Ascospore concentrations were noticeably similar between these sites, with peaks

occurring at approximately the same dates during 2011 and 2012. Weather parameters in

Winnipeg and Carman including daily mean temperature, daily mean relative humidity

and daily mean wind speed were similar throughout the season in 2011 and 2012.

Comparisons among total daily solar radiation in Winnipeg and Carman showed

similarities during 2011, but were different in 2012. Precipitation occurred on similar

days in Winnipeg and Carman during both years. Higher relative humidity and lower

temperatures were apparent in Carman during both years compared to Winnipeg.

Increases in atmospheric relative humidity tended to occur during precipitation events in

all site years.

46

Figure 2.4 Comparison of average daily mean ascospore concentrations over the

sampling period in misted (dashed lines) and non-misted (solid lines) plots in

Winnipeg (green lines) and Carman (blue lines) in A) 2011 and B) 2012.

47

Figure 2.5 Seasonal comparisons between Winnipeg (green lines) and Carman (blue

lines) in I) 2011 and II) 2012 among A) precipitation (mm) (bars), relative humidity

(%) (solid lines), B) temperature (°C) (solid lines), solar radiation (kw/m2) (dashed

lines) and C) wind speeds (m/s) (solid lines).

48

2.4.2.2 Precipitation and Misting Effects

Cumulative precipitation and ascospore concentrations during the flowering

period were closely matched based on the site-year (Figure 2.6). Precipitation and

ascopsore concentrations were not cumulated in Winnipeg during 2012 due to missing

data at the beginning of flowering. In Carman during 2011, accumulation of ascospore

concentrations and precipitation appear to be somewhat correlated. The 2012 ascospore

concentrations and precipitation accumulations were both slightly higher at the end of

flowering than those for Carman 2011. Carman 2012 had the highest accumulated

ascospore concentrations and precipitation totals overall at the end of the flowering

period.

In 2011, in Carman especially, major precipitation events occurred just prior to an

increase in ascospore concentration above the canopy (Figure 2.7). In Winnipeg (2011),

only a few precipitation events occurred during the ascospore sampling period whereas

during two events, peak ascsopore concentrations were reached. Another large

precipitation event occurred where spore concentrations are unknown. In Winnipeg in

2012, increases in ascospore concentrations followed precipitation events, with some

peaks occurring one day after rain. Carman 2012 also showed peaks following most

precipitation events, with some peaks occurring during a precipitation event. During

2011, misted plots did not show large differences in ascospore concentrations compared

to the non-misted plots. There appears to be higher concentrations in non-misted plots in

Carman and slightly higher concentrations among misted plots in Winnipeg. Peaks in

ascospore concentration occurred at similar times between misted and non-misted plots.

49

Figure 2.6 Cumulative mean daily ascospore concentrations (red) and total daily

precipitation (purple) from the 1st to the 24

th day of flowering in Carman 2011

(dotted line), Winnipeg 2012 (solid line) and Carman 2012 (dashed line).

50

Figure 2.7 Mean daily ascospore concentrations in ascospores per m

3 in A) Carman 2011

in misted (black lines) and non-misted (grey lines) plots B) Winnipeg 2011 in

misted (black lines) and non-misted (grey lines) plots C) Carman 2012 in non-

misted (grey lines) plots and D) Winnipeg 2012 in non-misted (grey lines) plots

plotted with total daily rainfall in mm (black bars) in A) Carman 2011 B) Winnipeg

2011 C) Carman 2012 and D) Winnipeg 2012. The flowering period is represented

by the time period between the * symbols.

*

51

2.4.2.3 Temperature, Relative Humidity, Solar Radiation and Wind Speed Effects

In all four site years, the majority of ascospore concentration peaks were either

preceded by peaks in relative humidity or occur simultaneously (Figure 2.8). However

these trends did not occur consistently throughout the sampling periods. Winnipeg 2011

tended to show increasing ascospore concentrations with increased relative humidity

during most peaks. Mean daily temperatures tended to increase during days with

decreasing relative humidity in the majority of cases. Thus, increases in temperatures

tended to occur simultaneously with peaks in ascospore concentrations but, again these

trends were not consistent. No trends were apparent among daily mean wind speed and

daily mean ascospore concentrations in all site years (Figure 2.9). Similarly, total daily

solar radiation and daily mean ascospore concentrations showed no apparent correlation.

During 2011, higher spore releases occurred on days with mean temperatures

between 18-22 °C in Carman (Figure 2.10). In Winnipeg, concentrations were higher on

days with mean daily temperatures from 14 to 18 °C. During 2012, highest ascospore

releases occurred on days with mean temperatures between 18-20 °C in Carman and 20-

22 and 26-28 °C in Winnipeg. Overall, highest ascospore releases occurred between 18 to

22 °C. Ascospores were released during daily mean relative humidity between 50 to 90%.

Highest concentrations were released from 74-78% and 62-66% mean daily relative

humidity in Winnipeg during 2011 and 2012 respectively with an overall range of

concentrations occurring between 50 to 78% in Winnipeg. In Carman, highest ascospore

concentrations occurred at daily mean relative humidity of 74-78 % in 2011 and 78-82%

52

in 2012 and all concentrations were within the range from 58 to 90% mean daily relative

humidity. Overall, highest daily ascospore concentrations occurred approximately from

62 to 82% mean daily relative humidity. In Winnipeg, highest values occurred during

days with 30 to 35% and 40-45% minimum relative humidity in 2011 and 2012

respectively. In Carman, days with minimum relative humidity at 50 to 55% and 60 to

65% achieved the highest ascospore concentrations during 2011 and 2012 respectively.

Days that experienced greater than a 20 percent decrease in relative humidity from 8 am

to 2 pm showed highest ascospore concentrations overall. Low daily mean ascospore

concentrations were experienced during days with increases below 20 percent. During

days where no precipitation occurred, daily mean ascospore concentrations were highest

overall, especially during 2012. Very little ascospore release occurred during large

precipitation events exceeding 6mm/ day. Overall, ascospore concentrations were highest

during days with mean wind speeds between 0.5 to 3.0 m/s and at wind speeds above 3

m/s ascospores release was either very low or spores were not captured in the device.

53

15

25

35

0

2000

4000

05-Jul 10-Jul 15-Jul 20-Jul 25-Jul 30-Jul 04-Aug 09-Aug 14-Aug

Air

Tem

pe

rature

(d

egre

es

celsiu

s)

Asc

osp

ore

C

on

cen

trat

ion

(sp

ore

s/m

3 )

*

60

80

100

0

2000

4000

05-Jul 11-Jul 17-Jul 23-Jul 29-Jul 04-Aug 10-Aug 16-Aug

Re

lative

Hu

mid

ity (%) A

sco

spo

re

Co

nce

ntr

atio

n

(sp

ore

s/m

3 )

II)A


3 (red lines) plotted

with average daily I) temperatures in degrees celsius (purple lines) and II) relative

humidity in percentage (purple lines) for A) Carman 2011 B) Winnipeg 2011 C)

Carman 2012 and D) Winnipeg 2012. The flowering period is indicated by the area

within the * symbols.

* I)A

I)B

I)C

I)D

54


3 (red lines) plotted

with I) mean daily wind speed (purple lines) and II) total daily solar radiation

(purple lines) for A) Carman 2011 B) Winnipeg 2011 C) Carman 2012 and D)

Winnipeg 2012.

55

Figure 2.10 Average of daily mean ascospore concentrations for non-misted plots in

Carman 2011, Winnipeg 2011, Carman 2012 and Winnipeg 2012 occurring within

specified intervals for factors including A) mean daily air temperature (°C), B)

mean daily relative humidity (%), C) minimum daily relative humidity (%), D)

daily decrease in relative humidity from 8am to 2pm (%) E) total daily precipitation

(mm) and F) mean daily wind speed (m/s).

56

2.4.3 Weather Impacts on Disease Incidence

According to the daily values computed for daily mean temperature, daily mean

relative humidity and total daily precipitation, it is evident that during a precipitation

event relative humidity increases slightly and temperatures decrease slightly. During

2011, little precipitation occurred during the period just prior to harvest when disease was

assessed in both Carman and Winnipeg. Winnipeg received slightly more rain at harvest,

however disease incidence at harvest was lower than Carman. During 2012, more

precipitation occurred before harvest, especially in Carman and percentage of infection

was approximately 15%. In Winnipeg, percentage of infection was low at harvest

followed by a small precipitation event. A large precipitation event occurred after harvest

and an increase in percentage of infection was determined post-harvest.

57

Figure 2.11. Percent infection counts during harvest (black diamond) and after harvest

(grey diamond) in A) Carman 2011, B) Winnipeg 2011, C) Carman 2012 and D)

Winnipeg 2012. Weather parameters include total daily precipitation in mm (black

bars), temperature in °C (grey lines) and relative humidity in % (black lines).

58

2.4.4 Misting and Seeding Rate Treatment Effects on the Canola Canopy

2.4.4.1 Canopy Density

Based on three different seeding rates and fertilizer requirements, three canopy

densities resulted. In 2011, at both locations there appears to be a difference between

high, medium and low seeding rates as represented by the LAI values in each of the 18

plots, although the distinction is more apparent at the Winnipeg location (Figure 2.12). In

2012, there was a clear difference between high and low seeding rates and a slight

difference between high, medium and low seeding rates at both locations. In 2011, from

budding to the end of flowering, a decreasing trend in LAI in Winnipeg was apparent,

although this trend did not occur in Carman where LAI increased. In 2012, no trends were

apparent in the Winnipeg plot due to the LAI sampling period which began just prior to

flowering to nearly the end of flowering. From the rosette stage to the end of flowering in

Carman (2012), an increasing trend was apparent.

The ANOVA for LAI revealed significant differences among all high and low

plots, with highest LAI under high seeding rate treatments (Table 2.6). In Winnipeg

during both years, high versus medium, high versus low and medium versus low plots

contained significantly different LAI. In 2011, Winnipeg contained significantly higher

LAI than Carman while in 2012, LAI values were not significantly different among

locations. No significant differences in LAI were apparent between years. According to

the ANOVA, plant counts were significantly lower in low versus high and low versus

medium plots in all site-years (Table 2.6). No significant differences in plant count

occurred among high and medium plots in any year except for Winnipeg 2011. Plant

counts were significantly higher in Winnipeg in 2011 and in Carman in 2012. Plant

59

counts in 2011 were significantly higher than 2012 overall. Distinguishable canopy

densities were also apparent in the field in both years, with plots seeded at high rates

being visibly dense, and plots appearing sparse at low seeding rates.

The ANOVA results for misting and seeding rate treatments on LAI revealed

significant differences in Winnipeg for high versus medium, high versus low and medium

versus low seeding rate treatments in both misted and non-misted plots (Table 2.7). In

Carman, high seeding rate treatment plots contained significantly higher LAI than low

plots and higher LAI under medium versus low plots under both misted and non-misted

plots. LAI and plant counts were significantly higher overall in Carman compared to

Winnipeg. Significantly higher plant counts occurred in misted plots in Carman, with no

significant effects of misting in Winnipeg (Table 2.7). High seeding rate treatments

yielded significantly higher plant counts compared to low seeding rates treatments in

Carman, while in Winnipeg high versus low and medium versus low seeding rate plots

showed significant differences. There were significant differences among LAI and plant

counts in Winnipeg and Carman where both misted and non-misted plots were included

in the ANOVA. Combined effects of misting treatments and seeding rate treatments did

not significantly affect LAI or plant counts at either location.

60

Figure 2.12 Leaf area index values taken from plots 1-18 throughout the A) 2011

growing season in Carman (plots 1-9 are misted) B) 2011 growing season in

Winnipeg (plots 10-18 are misted) C) 2012 growing season in Carman and D) 2012

growing season in Winnipeg. Blue, green and dotted yellow lines represent plots

seeded at high, medium and low seeding rates respectively.

A

B

B

C

D

61

Table 2.6 Significance levels from analysis of variance on the effects of seeding rate on

leaf area index (LAI) and plant counts from Winnipeg and Carman in 2011 and

2012 under natural weather conditions (non-misted plots). Words in brackets

indicate which selection (year or location) is higher.

Winnipeg 2011 Carman

2011 Winnipeg 2012 Carman

2012 Non-misted Non-misted Non-misted Non-

misted Effects Pr>f Pr>f Pr>f Pr>f

Year on LAI 0.5998 (2012)

Location on LAI 0.0142 (Wpg) 0.8884 (Wpg)

Seed Rate on LAI <.0001 <.0001 <.0001 <.0001

High vs. Low <.0001 <.0001 <.0001 <.0001

High vs. Medium <.0001 0.1667 <.0001 0.0074

Medium vs. Low <.0001 0.0001 0.0026 <.0001

Year on Plant Count 0.0028 (2011)

Location on Plant Count <.0001 (Wpg) <.0001 (Car)

Seed Rate on Plant Count <.0001 0.0148 0.0017 0.0001

High vs. Low <.0001 0.0088 0.0005 <.0001

High vs. Medium 0.0371 0.8261 0.2037 0.1118

Medium vs. Low 0.0068 0.0153 0.0162 0.0044

Table 2.7 Significance levels from analysis of variance on the effects of seeding rate and

misting on leaf area index (LAI) and plant counts in Winnipeg and Carman in 2011.

Words in brackets indicate which selection (location, misting) is higher.

Winnipeg 2011 Carman 2011

Misted Non-misted Misted Non-misted

Effects Pr>f Pr>f Pr>f Pr>f

Location on LAI <.0001 (Car)

Misting on LAI 0.1020 (Mist) 0.2318 (NonMist)

Seed Rate on LAI <.0001 <.0001

Seed Rate on LAI <.0001 <.0001 <.0001 <.0001

High vs. Low <.0001 <.0001 <.0001 <.0001

High vs. Medium <.0001 <.0001 0.1631 0.0025

Medium vs. Low <.0001 <.0001 0.0002 0.0009

Misting*Seeding Rate on LAI 0.6817 0.5811

Location on Plant Count <.0001 (Wpg)

Misting on Plant Count 0.2475 (NonMist) 0.0295 (Mist)

Seeding Rate on Plant Count 0.0023 0.0036

Seed Rate on Plant Count 0.0202 0.0027 0.0059 0.0784

High vs. Low 0.0007 0.0080 0.0424 0.0016

High vs. Medium 0.1062 0.5254 0.8646 0.0481

Medium vs. Low 0.0363 0.0340 0.0604 0.1519

Misting*Seeding Rate on Plant

Count 0.8209 0.0598

62

2.4.4.2 Canola Microclimate

Several observations can be made based on the test for analysis of variance

(ANOVA) on microclimatic parameters under all three treatments for non-misted

conditions in 2011 and 2012 (Table 2.8). In 2011, there were significantly lower relative

humidity, temperatures and leaf wetness periods. In Carman, relative humidity was

significantly higher during both site years and as a result leaf wetness was also higher.

Temperatures were significantly lower in Carman compared to Winnipeg in 2011.

Significant differences occurred between high versus low seeding rate treatments with

respect to relative humidity and temperature during three site-years including Winnipeg

2011, Carman 2012 and Winnipeg 2012. In these cases, temperatures were higher among

low density plots and relative humidity was higher under high density plots. Soil

temperatures were significantly higher in low versus high density plots at both sites in

both years and significantly higher values were observed in medium versus low plots in

all but the Carman 2011 location.

Misted plots contained significantly higher relative humidity and leaf wetness

values, but lower temperatures (Table 2.9). Soil temperatures were not significantly

affected by misting. Relative humidity, temperature, as well as leaf wetness parameters

were significantly different among high and low plots in most site-years. Thus, misting

and variable seeding rates did create significant difference for all microclimate

parameters.

63


microclimatic parameters including relative humidity (RH) in %, temperature (T) in

°C, growing degree days (GDD) in days and leaf wetness (LW) from Winnipeg and

Carman in 2011 and 2012 under natural weather conditions (non-misted plots)

during the flowering period. Words in brackets indicate which selection (year or

location) is higher.

Winnipeg

2011

Carman

2011

Winnipeg

2012

Carman

2012 Non-misted Non-misted Non-misted Non-misted


Year on RH 0.0053 (2012)

Location on RH <.0001 (Car) 0.0003 (Car)

Seed Rate on RH <.0001 0.1248 0.0006 0.0001

High vs. Low <.0001 0.0456 0.0002 <.0001

High vs. Medium 0.0349 0.4583 0.1330 0.4392

Medium vs. Low 0.0002 0.1923 0.0098 0.0007

Year on T <.0001 (2012)

Location on T 0.0002 (Wpg) 0.2023 (Wpg)

Seed Rate on T 0.0057 0.5068 0.0081 0.0011

High vs. Low 0.0016 0.2831 0.0021 0.0007

High vs. Medium 0.0980 0.8703 0.1080 0.7362

Medium vs. Low 0.0532 0.3605 0.0800 0.0018

Year on LW 0.1135 (2011)

Location on LW 0.0004 (Car) <.0001 (Car)

Seed Rate on LW 0.8387 0.6229 0.8993 0.1916

High vs. Low 0.6684 0.3379 0.6531 0.2964

High vs. Medium 0.8922 0.6930 0.7691 0.4275

Medium vs. Low 0.5736 0.5688 0.8755 0.0726

Year on Soil T 0.0010 (2012)

Location on Soil T 0.0656 (Car) 0.0082 (Car)

Seed Rate on Soil T 0.0006 0.1212 0.0053 0.0192

High vs. Low <.0001 0.0024 0.0004 0.0086

High vs. Medium 0.0674 0.0903 0.0815 0.8130

Medium vs. Low 0.0005 0.1283 0.0301 0.0153

64

Table 2.9 Levels of significance from analysis of variance on the effects of seeding rate

treatment and misting on microclimate parameters in Winnipeg and Carman in 2011

during the flowering period. Words in brackets indicate which selection misting or

location) is higher.




Location on RH <.0001 (Car)

Misting on RH 0.0003 (Mist) 0.0003 (Mist)

Seed Rate on RH <.0001 0.0041

Seed Rate on RH <.0001 <.0001 0.0424 0.0477

Low vs. High <.0001 <.0001 0.0158 0.0206

Medium vs. High 0.0045 0.0111 0.3583 0.7386

Medium vs. Low <.0001 <.0001 0.1095 0.0426

Misting x Seeding Rate on

RH

<.0001 0.0006

Location on T <.0001 (Wpg)

Misting on T 0.0002 (NonMist) 0.0013 (NonMist)

Seed Rate on T <.0001 0.0003

Seed Rate on T <.0001 <.0001 0.0020 0.0375

Low vs. High <.0001 <.0001 0.0176 0.0006

Medium vs. High 0.0003 0.0038 0.7038 0.2556

Medium vs. Low <.0001 <.0001 0.0405 0.0106

Misting x Seeding Rate on T <.0001 0.0003

Location on Leaf Wetness <.0001 (Car)

Misting on Leaf Wetness <.0001 (Mist) <.0001 (Mist)

Seed Rate on Leaf Wetness 0.0006 0.0006

Seed Rate on Leaf Wetness 0.0003 0.2468 0.0021 0.0308

Low vs. High 0.2219 <.0001 0.0093 0.0102

Medium vs. High 0.6956 0.2427 0.2591 0.2799

Medium vs. Low 0.1119 0.0019 0.1077 0.0007


Leaf Wetness

<.0001 <.0001

Location on Soil T 0.3356 (Car)

Misting on Soil T 0.9989 (NonMist) 0.1180 (NonMist)

Seed Rate on Soil T <.0001 0.0005

Seed Rate on Soil T <.0001 0.0002 0.0045 0.0315

Low vs. High 0.0019 0.0023 0.0415 0.2288

Medium vs. High 0.9958 0.8851 0.7313 0.9281

Medium vs. Low 0.0926 0.0926 0.8221 0.9653


Soil T

<.0001 0.0018

65

2.4.4.3 Microclimate and Disease Development

Canopy density, disease development and microclimatic variable comparisons

among the 4 site-years are shown in Table 2.10. LAI values were comparable among all

site years, with the highest values occurring in Carman (2011). Highest plant counts were

observed in Winnipeg in 2011, with the lowest counts in Winnipeg in 2012. Carman

contained higher plant counts in 2012 compared to 2011. With respect to disease, mean

ascospore concentrations were highest in Carman (2012). Percentages of infection at

harvest were highest in 2011, with higher infection percentages in Carman than Winnipeg

in both years. Carman also experienced higher within canopy leaf wetness and relative

humidity values and lower air temperature values during both years. In 2011, relative

humidity and leaf wetness were higher and temperatures were lower under the canopy

compared to 2012.

Misted plots in Winnipeg 2011 showed higher overall LAI and lower plant counts,

but higher plant counts and lower LAI values were recorded in Carman 2011 (Table

2.10). Both ascospore concentrations and percentages of infection were higher among

misted plots in Winnipeg 2011 than non misted plots, however in Carman 2011 the non-

misted plots contained higher values than misted plots. Significant differences were

apparent among microclimates in misted plots at both locations with increased values for

relative humidity and leaf wetness, and decreased air and soil temperatures.

66

Table 2.10 Summary of whole plot mean ascospore concentrations (ascospores/m3),

disease incidences (%) and microclimate parameters. 2011 misted and non-misted

mean ascospore concentrations, disease incidences and microclimatic parameters

are listed. Data was used only for days where all values are present and for over

lapping Winnipeg and Carman days only.

Winnipeg Carman

Variable Misted Non-Misted All Misted Non-Misted All

2011

Leaf Area Index 3.2 3.0 3.1 3.4 3.5 3.5

Plant Counts (plants/m3) 133.1 148.3 140.7 107.2 77.5 92.4

Ascospore Concentration

(ascospores/m3) 705 628 671 569 663 616

Percent Infection (%) 6.0 5.5 5.8 20.7 22.5 21.6

Relative Humidity (%)x 78.2 76.6 77.4 84 82.2 83.1

Leaf Wetnessx 32.9 24.4 28.7 47.8 41 44.4

Air Temperature (°C)x 20.6 20.8 20.7 20 20.4 20.2

Soil Temperature (°C)x 20.4 20.4 20.4 20.4 21.2 20.8

2012

Leaf Area Index 3.1 3.1 3.0 3.0

Plant Counts (plants/m3) 75.9 75.9 111.0 111.0

Ascospore Concentration

(ascospores/m3) 694 694 2170 2170

PercentInfection (%)

3z, 9

y 3

z, 9

y

16 16

Relative Humidity (%)x 76.3 76.3 80.7 80.7

Leaf Wetnessx 25.9 25.9 39.1 39.1

Air Temperature (°C)x 21.9 21.9 21.1 21.1

Soil Temperature (°C)x 21.3 21.3 20.9 20.9

z Percentf Infection was counted at harvest y Percent Infection was counted post-harvest xData obtained from 4th leaf to harvest

67

2.4.4.4 Ascospore Concentrations and Disease Incidence

No significant impacts (p > 0.05) were found on ascospore concentrations among

the three seeding rate treatments in any site-year (Table 2.11). Significantly higher

ascospore concentrations occurred in 2012 overall, with highest concentrations in

Carman. There was significantly higher disease incidence in 2011 overall. At Carman,

there was significantly higher infection than in Winnipeg during both years. The only

significant differences in percentage infection occurred between high and low seeding

rate treatments in Carman in 2011, where high seeding rates produced more disease. Total

daily mean ascospore concentrations and disease levels were most affected by seeding

rate as shown in Figures 2.13 and 2.14 with the exception of Carman 2011 where seeding

rate treatments appeared to influence percentage of infection (Figure 2.14).

A comparison of the accumulated ascospore concentrations between high,

medium and low density plots showed no consistent differences among the three

treatments (Figure 2.13). In the Carman 2011 and Winnipeg 2012 plots, accumulated

daily mean ascospore concentrations at the end of flowering are highest among high

density plots and lowest among medium density plots. In Carman 2012, accumulated

daily mean ascospore concentrations are highest among low density plots and lowest

among medium density plots. Overall, the 2012 sites showed higher accumulated daily

mean ascospore concentrations by the end of the flowering period compared to the 2011

Carman site. From 15 to 20 days after flowering, Carman 2012 showed a steep rise in

ascospore concentration which did not occur during the other 2 site-years.

68

Average daily mean ascospore concentrations and percentage of infection were

analyzed under misted versus non misted conditions in 2011 (Table 2.12). No significant

effects of location, misting or seeding rates were seen among average daily mean

ascospore concentrations. With respect to infection percentages, Carman produced

significantly higher percentages than Winnipeg. High and low seeding rate treatments

showed significantly different disease percentages in both misted and non-misted plots in

Carman; no significant differences were observed in Winnipeg among seeding rate

treatments. Misting also had no effect on percent sclerotinia infection at either location.

69


average daily mean ascospore concentrations and disease incidence (DI) from

Winnipeg and Carman in 2011 and 2012 under natural weather conditions (non-

misted plots). Words in brackets indicate which selection (year or location) is

higher.

Winnipeg 2011 Carman 2011 Winnipeg 2012 Carman 2012

Non-misted Non-misted Non-misted Non-misted


Year on Spore Concentration <.0001 (2012)

Location on Spore Concentration 0.1220 (Car) <.0001 (Car)

Seed Rate on Spore Concentration 0.9146 0.5872 0.9660 0.2494

High vs. Low 0.7669 0.5230 0.8106 0.4567

High vs. Medium 0.6805 0.7040 0.8344 0.4036

Medium vs. Low 0.8671 0.3115 0.9756 0.0988

Year on DI 0.0011 (2011)

Location on DI <.0001 (Car) <.0001 (Car)

Seed Rate on DI 0.8651 <.0001 0.6004 0.6969

High vs. Low 0.7296 <.0001 0.6446 0.3437

High vs. Medium 0.8563 0.0030 0.3996 0.8514

Medium vs. Low 0.5989 0.0146 0.7006 0.4457

Table 2.12 Significance levels from analysis of variance on the effects of seeding rate

and misting on average daily mean ascospore concentrations (Spore Concentration)

and disease incidence (DI) in Winnipeg and Carman in 2011. Words in brackets

indicate which selection (location, misting) is higher.




Location on Spore Concentration 0.5459 (Wpg) Misting on Spore Concentration 0.1432 (Mist) 0.4875 (NonMist)

Seed Rate on Spore Concentration 0.9946 0.8138

Seed Rate on Spore Concentration 0.5030 0.6824 0.9133 0.4435 High vs. Low 0.5744 0.4362 0.4285 0.9706

High vs. Medium 0.3912 0.2564 0.6368 0.7198 Medium vs. Low 0.7015 0.7129 0.2123 0.6954

Misting*Seeding Rate on Spore Concentration 0.4864 0.7770

Location on DI <.0001 (Car)

Misting on DI 0.8939 (Mist) 0.5866 (NonMist)

Seeding Rate on DI 0.9928 0.0006

Seed Rate on DI 0.8840 0.9338 0.0676 0.0023 High vs. Low 0.8129 0.8339 0.0006 0.0229

High vs. Medium 0.9013 0.7787 0.0380 0.3766 Medium vs. Low 0.7186 0.6243 0.0898 0.1390

Misting*Seeding Rate on DI 0.9946 0.0054 (NonMist,High)

70

Figure 2.13 Cumulative ascospore concentrations (ascospores/m3) in high (blue),

medium (green) and low (yellow) plots in Carman 2011, Winnipeg 2012 and

Carman 2012.

Carman 2012

Winnipeg 2012

71

Values obtained for mean LAI across the growing season and plant counts in each

plot were plotted against percentages of infection (Figure 2.14) and ascospore

concentration (Figure 2.15). The coefficients of determination obtained from these

correlations indicate that ascospore concentration is not associated with LAI or plant

counts during any of the four site-years. Percentage of infection was weakly associated to

LAI and plant count in Carman in 2011; however no other site years showed this

association..

72

Figure 2.14 Relationship between sclerotinia stem rot disease incidence (%) and a) LAI;

b) plant density in Winnipeg 2011 (black line), Carman 2011 (grey line), Winnipeg

2012 (dotted black line) and Carman 2012 (dotted grey line).

73

Figure 2.15 Relationship between ascospore concentration (ascospores/m

3) and a) LAI;

b) plant density in Winnipeg 2011 (black line), Carman 2011 (grey line), Winnipeg

2012 (dotted black line) and Carman 2012 (dotted grey line).

74

Correlation between total ascospore concentrations (excluding data gaps) and

disease incidence were calculated for Winnipeg and Carman. The regression (R2) between

all total ascospore concentrations (ascospores/m3) and disease incidences (%) for each

canola plot showed no linear relationship between the two variables for either Winnipeg

or Carman (R2 = 0.0314 and 0.0115 respectively). Similarly, in Winnipeg no linear

relationships were observed within each of the high, medium and low treatments

comparing disease incidence (%) to total ascospore concentration with R2 values of

0.0076, 0.1041 and 0.0512 respectively. No linear relationships were observed in Carman

among the three treatments with R2

values of 0.6133 (negative), 0.0867 and 0.066

(negative) in high, medium and low treatments respectively.

2.4.4.6 Microclimate Time Series Comparisons to Daily Ascospore Production

Figures 2.16 through 2.19 show no clear correlation between microclimate

parameters and ascospore release. Among environmental factors, peaks in relative

humidity coincide with peaks in leaf wetness and increasing temperatures within the

canopy also lead to increased soil temperatures. Occasionally, peaks in daily mean

ascospore concentrations lag peaks in relative humidity.

75

Figure 2.16 Comparisons of A) ascospore concentrations (ascospores/m3) and overall

microclimatic averages in high, medium and low plots for B) daily air temperature

(˚C), C) daily relative humidity (%), D) daily soil temperature (˚C) and E) daily leaf

wetness in Carman, 2011.

76




wetness in Winnipeg, 2011.

77




wetness in Carman, 2012.

78




wetness in Winnipeg, 2012.

79

2.4.4.5 Petal Test Results

In 2011, petal tests were done in Carman and Winnipeg at 30 and 70 percent

bloom stages to ensure the presence of ascospores on petals in each field. In 2012, petal

tests were done in Carman and Winnipeg at 15, 30 and 70 percent bloom stages in each of

the plots. Based on the observations made approximately 14 days after plating, no

consistent results were found (Table 2.13). Very few sampled petals contained ascospores

during any of the sampling periods.

Table 2.13. Petal counts containing ascospores within each plot at various bloom stages.

Non listed plots contained 0 ascospore colonized petals.

Year Location Date Bloom

Stage (%) Plot # # Petals with ascospores

2011

Winnipeg

Jul-06 30 18 1

3 1

Jul-14 70 12 1

13 4

14 1

Carman Carman

Jul-18 30 8 2

Jul-25 70 None 0 2012 Winnipeg

Jul-13 15 17 3

5 1

Jul-19 30 14 1

11 1

Jul-24 70 None 0 Carman Jul-05 15 2 2

3 1

Jul-09 30 14 1

Jul-18 70 12 1

80

2.5 Discussion

2.5.1 Weather and Disease Development

This study suggests that a higher concentration of inoculum in the form of

sclerotia may have been responsible for higher ascospore concentrations in Carman

compared to Winnipeg in 2011. In comparing ascospore concentrations between

Winnipeg and Carman where equal inoculum was provided to both sites in 2012, higher

rates were released in Carman due to the elevated relative humidity, reduced temperatures

and additional precipitation. Evidently weather plays a major role in ascospore production

due to the similarity in peak spore concentrations between Winnipeg and Carman situated

more than 50 km apart. During late bloom, precipitation led to increased airborne

ascospore concentrations. Peaks in ascospore concentrations generally followed

prolonged periods of increased relative humidity following a precipitation event, similar

to observations by Qandah and Mendoza (2011). Higher precipitation rates in Carman

during 2012 also correlated to increased infection towards harvest. Decreased temperature

along with increased relative humidity and leaf wetness appear to be responsible for

higher disease incidence in Carman compared to Winnipeg. Canopy density

modifications effectively altered microclimates however, these changes were not

sufficient to significantly influence ascospore release and infection. Similarly, disease

development was not significantly affected by misting despite the creation of a cooler and

moister canopy.

81

Specific environmental conditions are required for crop and disease development.

Crops require temperatures, moisture and nutrients within a given range for survival and

growth. Similarly, sclerotinia stem rot is increased under specific weather conditions. For

example adequate moisture and cool temperatures have been shown to be required for

disease development (Turkington et al., 1991; Manitoba Agriculture, 2013). The first year

of this study, 2011, began with seeding occurring later than normal throughout Manitoba

in 2011 due to the saturated top soils, heavy snow melt and precipitation during May and

June. The rest of the summer was fairly dry and hot compared to normal. Warm dry

weather in April preceded a prolonged cool period at the beginning of May in 2012

followed by warm dry conditions during the summer. Slightly warmer temperatures and

lower relative humidity were experienced in 2012 reducing time to flowering and

affecting seed set and size. Microclimates were also affected by the overall weather

creating warmer temperatures and reduced relative humidity below the canopy in 2012.

Some interesting trends were observed among all the variables compared among both

years and sites. With respect to canopy density, site years with higher LAI values did not

contain the highest plant counts. For example, Winnipeg 2011 showed the highest LAI,

however plant counts were below average. One of the reasons for this may be due to the

presence of weeds which were competing with canola for available resources and

covering additional surface area thus increasing LAI values and decreasing plant counts.

Weeds were also abundant in Winnipeg in 2012. Higher LAI measurements generally

during early bloom can be explained by the thinning of the canopy from early to full

flowering to pod development.

82

With respect to disease, mean ascospore concentrations were higher in 2012 which

may have resulted from the additional inoculum levels in the form of sclerotia placed on

the fields that year. However, the increased ascospore concentrations released from

apothecia did not result in increased infection. Thus environmental factors influencing

disease incidence must have been a limiting factor. Additionally, average, minimum and

maximum mean daily ascospore concentrations were higher during late bloom compared

to early bloom. This trend may have occurred as a result of the natural timing of

ascospore release occurring during the growing season or due to weather conditions

during late bloom favoring ascospore release from apothecia. For species Leptosphaeria

maculans (the pathogen responsible for blackleg in canola), humid conditions with

frequent rainfall and prolonged moisture favour ascospore release with air temperatures at

approximately 13 to 18 C and relative humidity values above 80 per cent (Canola Council

of Canada, 2014). Leptosphaeria maculans ascospore dispersal occurs several hours after

a precipitation event of greater than 2 mm and can persist for approximately 3 days (Guo

and Fernando, 2005). Similarly, the ascospores discharged into the atmosphere by

Fusarium graminearum (fusarium head blight in wheat) require warm (25 to 28 C) moist

conditions during flowering for successful infection (Government of Saskatchewan,

2007). Precipitation may have also influenced S. sclerotiorum ascospore release in several

ways in this study. Due to different seeding dates among site years, precipitation was

variable throughout the growing season where totals were higher in Winnipeg in 2011

and Carman in 2012. Seasonal precipitation was higher under sites with earlier seeding

dates. The high precipitation in Carman (2012) increased spore concentrations

significantly and also resulted in high disease incidence. In 2012, more precipitation was

received overall during the sampling period which may also have contributed to increased

83

spore concentrations; however overall disease incidence at harvest was not affected.

Precipitation was increased during late bloom compared to early bloom which may

account for higher ascospore concentrations that occurred during late bloom.

Accumulated total precipitation seems to have influenced accumulated ascospore release

throughout the flowering period since highest precipitation totals led to higher

accumulated ascospore concentrations. Average daily mean relative humidity values that

were higher in Carman compared to Winnipeg during both years may be partially

responsible for the increased disease and ascospore concentrations occurring in Carman.

Several researchers (Tores and Moreno, 1991; Clarkson et al, 2003; Qandah and

Mendoza, 2011) have discussed the influence of not only relative humidity on SSR

disease development but other weather factors such as temperature, solar radiation and

wind speed. The field in Winnipeg in 2012 was located within 30 meters of a fence with

trees. These obstructions may be the reason why average daily wind speeds at this

location were low in comparison to Carman. Winnipeg 2012 also experienced very low

infection levels; however the influence of wind on disease development is not clear.

Temperatures were highest during the Winnipeg 2012 growing season compared to other

site years which may also have contributed to a reduction in disease. Based on previous

research, sclerotia germinate most readily at an optimum temperature of 10°C (Abawi and

Grogan, 1975) while ascospore release is optimum at 22°C according to Qandah and

Mendoza (2011). Ascospore survival is also important for disease occurrence; survival

rates at temperatures at or above 25°C are reduced (Caesar and Pearson, 1983). Koch et

al. (2007) showed that most rapid SSR development occurred between 16 and 22°C.

During both years at late bloom, Winnipeg showed higher values for average total daily

84

incoming solar radiation compared to Carman. This may have increased temperatures late

in the season and possibly reduced the potential for disease incidence.

Comparisons of ascospore concentrations taken during the sampling period between

Winnipeg and Carman plots show similar trends with peaks occurring on the same days.

Since values for daily weather parameters plotted for Winnipeg and Carman during the

season also show similar trends, we can conclude that daily ascospore release over time

was responding to weather conditions. It is evident that with precipitation events, there is

an increase in daily mean percent relative humidity percentages. In a study conducted by

Qandah and Mendoza (2011), peaks in sclerotinia ascospore concentrations occurred after

prolonged periods of high relative humidity followed by a rapid decrease in relative

humidity. The result of this study confirms their findings as similar trends were observed.

However, this pattern did not occur consistently and thus has limited value for prediction

of ascospore release. Peaks in daily mean ascospore concentrations generally occurred

following a precipitation event which would have caused an increase in atmospheric

relative humidity. The majority of higher relative humidity values occurred slightly

before or during increases in atmospheric ascospore concentrations. In most cases, when

relative humidity decreased and temperatures increased slightly, ascospore concentration

peaks were observed. These trends indicate that prolonged moisture and increased relative

humidity prior to ascospore release may be required; however release occurred during

decreasing relative humidity while temperatures were increasing and relative humidity

was decreasing. Although this phenomenon appears to have occurred during most peak

ascospore days, it is not the case throughout the sampling period. Other factors combined

with precipitation, relative humidity and temperature need to be examined.

85

Effects of precipitation and irrigation on sclerotinia stem rot has been studied by

several researchers (Blad et al., 1978; Twengstrom et al., 1998; Bom and Boland, 2000;

Qandah and Mendoza, 2011), however the effects of misting on S. sclerotiorum ascospore

release in canola remains uncertain. Natural and artificial precipitation contributes to

increased soil moisture, canopy wetness and relative humidity which are considered to be

suitable environmental conditions for SSR disease development. Precipitation often also

contributes to decreased temperatures under the canopy favoring disease slightly, as

shown by Blad et al. (1978) and Weiss et al. (1980) in their studies of irrigation on dry

edible beans. Soil moisture is responsible for germination of sclerotia, influencing an

important inoculum source (Twengstrom et al., 1998; Bom and Boland, 2000). Relative

humidity plays an important role in the release of ascospores from apothecia (Qandah and

Mendoza, 2011). Similar results of misting on microclimate were shown in this study.

The site-year containing the highest accumulated daily precipitation also showed the

highest accumulated mean daily ascospore concentration among all site-years. Thus, a

positive correlation between accumulated precipitation and ascospore concentration was

evident. During 2011 when plots were misted, Winnipeg showed higher daily mean

ascospore concentrations and percent infection on average under misted compared to non-

misted plots, however in Carman non-misted plots showed higher ascospore

concentrations and higher percentage of infection on average. In Winnipeg, misting may

have contributed to increased spore concentrations on average, but other unknown factors

may also be responsible. The plot in Carman contained a history of disease in the non-

misted portion of the field, therefore even with the addition of equal inoculum levels, the

non-misted portion still contained higher levels during the season. Although misting may

86

contribute slightly to ascospore release, level of inoculum present is also a critical factor

in determining ascospore release concentrations and overall disease incidence. There also

does not appear to be a combined effect of misting and precipitation on ascospore release

as trends in concentrations are similar among misted and non-misted plots. Additional

focus needs to be placed on the effects of misting on ascospore release where inoculum

levels are similar throughout treatments.

Ascospore release from apothecia is an important part of the disease life cycle as it

contributes to the source of inoculum present in the proximate atmosphere available for

colonization and infection of the canola crop. Several environmental factors such as

precipitation, relative humidity, temperature, wind and solar radiation may affect

inoculum levels. As previously mentioned, precipitation contributes to increased

atmospheric relative humidity; however ascospore release does not occur during large

precipitation events according to this study. Ascospore release may be linked to a series

of environmental changes occurring following a rain event as increases in ascospore

concentrations tend to occur during the days following rain. Ascospores of Leptosphaeria

maculans are also released following larger precipitation events (Guo and Fernando,

2005). Sclerotinia sclerotiorum ascospore release generally occurs mid-day as shown by

several researchers (Harthill, 1980; McCartney and Lacey, 1991). In a study conducted by

Qandah and Mendoza (2011), ascospore release generally occured in a single event daily

from 8 am to 4pm with highest ascospore concentrations occurring from 10am to 2pm.

The increase in ascospore release occurring from 8am to 2pm was reported to be a result

of increasing temperature and decreasing relative humidity (Qandah and Mendoza, 2011).

This study focuses further on these trends and tries to determine the optimum temperature

87

and relative humidity changes required to increase ascospore release assuming ascospore

release is occurring in a single event from 8 am to 4pm daily. Relative humidity

decreases and temperature increases from 8am to 2pm for each day were compared

against daily mean ascospore concentrations. This time period was chosen to represent

the period at which ascospore concentrations are said to be increasing during the day

(Qandah and Mendoza, 2011). In this study, relative humidity decreases computed for

each day from 8am to 2pm showed higher daily mean ascospore concentrations where

daily decreases exceeded 20%; below that, low concentrations were released among most

locations. This pattern may highlight the importance of rapidly decreasing relative

humidity required for spore release which is followed by a period of elevated relative

humidity. Increases in temperature from 8am to 2pm between 6 to 8°C tended to correlate

to highest ascospore release in Carman 2012, however no distinct trends were realized.

Daily mean values for several environmental factors were used in comparison to

ascospore concentrations. On average, days with mean temperatures between 18 to 22 °C

appear to have had the highest average mean daily ascospore release concentrations

indicating that ascospores tend to be released in high concentrations during days with

approximately average seasonal temperatures in Manitoba. Days with mean percent

relative humidity between 62 and 82% produced the highest mean daily ascospore

concentrations overall in all site years. Relative humidity in Carman was slightly higher

compared to Winnipeg during both years. Higher minimum and mean percent relative

humidity was required to increase ascospore release in Carman whereas ascospore

concentrations were higher under the increased range of relative humidity compared to

Winnipeg. This may be explained by an adaptive behavior of the pathogen which is

capable of adapting to regions with varying environments. Although lower relative

88

humidity in Winnipeg may be responsible for the reduced ascospore concentrations

overall, release of spores still occurred under these conditions. Lower daily mean wind

speeds ranging from 0.5 to 3.0 m/s contributed to the highest daily mean ascospore

concentrations. Slight winds may be required for the release and dispersal of ascospores,

especially to a height above the canopy. At higher daily mean wind speeds ascospore

concentrations were reduced which could reflect a damaging result of wind on apothecia

or the influence wind has on the mechanical ability of the spore capturing device. Higher

wind speeds may also remove ascospores from the boundary layer.

Disease incidence is one of the most important indicators of disease at harvest

measured as percent infection. It is understood that precipitation alters other atmospheric

parameters by increasing relative humidity and decreasing temperature. Combined with

the modifications associated with precipitation, they can enhance disease development

based on previously completed studies (Twengstrom et al., 1998; Bom and Boland, 2000;

Qandah and Mendoza, 2011). Based on observations, precipitation just prior to harvest

did not correlate with disease incidence between site years and therefore does not appear

to affect disease incidence significantly. Based on the 2011 data from both sites, slightly

higher precipitation prior to harvest in Winnipeg did not result in increased disease

incidence compared to Carman. However increased disease incidence in Carman may be

attributed to the disease history at this location with increased inoculum levels. In 2012

both fields received even inoculum levels and precipitation just prior to harvest were

similar which correlated to high percent of disease at harvest in Carman. In Winnipeg

(2012), a small precipitation event prior to harvest provided low disease percentage at

harvest. An increase in disease incidence occurring post-harvest may be in part due to the

89

larger rain event occurring before the assessment was complete. Other diseases such as F.

graminearum requires at least 12 hours of precipitation and high relative humidity for

successful germination and infection of ascospores (Government of Saskatchewan, 2007).

Another explanation could be that disease is also enhanced over time showing greater

symptoms and is easier to identify as it worsens. Similarly, percentage of diseased lettuce

plants increased over time in a study conducted by Young et al. (2004). Although

precipitation contributed to higher disease percentages in 2012, it does not appear to be a

limiting factor for disease incidence since disease incidence was more strongly correlated

to inoculum levels and ascospore release loads. It is possible that adequate moisture was

supplied to the crops in all site-years and other weather factors may have more influence

on disease incidence. Young et al. (2004) studied S. sclerotiorum disease development on

lettuce and found that disease development was not influenced by leaf wetness duration

or relative humidity; however disease developed quickly under optimum temperatures of

15 – 25 °C and continual moisture. Lower July and August daily minimum and maximum

temperatures are considered to enhance disease prevalence due to the “cool-climate

disease” label sclerotinia has acquired (Workneh and Xuang, 2000). During any given

year, maximum and minimum temperatures that were lower than the normal for a specific

region received increased disease prevalence (Workneh and Xuang, 2000). On average

during late bloom and ripening, mean, minimum and maximum temperatures were

highest within Winnipeg plots during both years and lower disease incidence percentages

were also observed in Winnipeg.

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2.5.2 Effect of Microclimate on Disease Development

Environmental conditions below the canopy are most important for crop and

disease development. Temperatures between 10 to 20 C (Phillips, 1987) and adequate soil

moisture (Matheroon and Porchas, 2005) are necessary for sclerotial germination.

Ascospore release from apothecia relies on prolonged periods of high relative humidity

and followed by decreasing relative humidity and increasing temperatures mid-day

(Qandah and Mendoza, 2011). Upon landing on senescent tissue, ascospores require

moisture in the form of leaf wetness to germinate and further infect the plant (Bardin and

Huang, 2001). In comparing the overall microclimatic conditions between all 4 site-years,

relative humidity and leaf wetness were higher and temperatures were reduced in Carman

during both years. Overall, 2012 experienced higher temperatures and lower relative

humidity and leaf wetness. Below canopy conditions reflect the weather conditions

above the canopy. It appears that the microclimatic conditions experienced in Carman

during both years were conducive to disease increasing the overall percentage of infection

during both years. These conditions may have also created favorable conditions for

ascospore release and dispersal based on the high ascospore concentrations observed in

Carman during 2012.

Increasing canopy density serves as an important tool for a variety of agricultural

improvements, however there may be drawbacks. Increasing canopy density generally

provides increased yields and reduces weed pressures though several canola diseases

including sclerotinia are shown to thrive under conditions created by denser canopies. A

combination of seeding rates and fertilizer treatments has been shown to be effective in

the modification of canopy density, microclimate factors, as well as disease severity.

91

Secondary spread of disease can be reduced in beans and sunflower by increasing row

spacing and reducing seeding rates (Hoes and Huang, 1985; Tu and Zheng, 1997). In

canola, conflicting results have been found on the effects of canopy density on disease

incidence. Turkington and Morall (1990) and Jurke and Fernando (2008) saw an

influence of canopy density on disease incidence. Lodging was more prevalent in high

density plots, creating conditions conducive to disease development such as increased

contact and moisture (Jurke and Fernando, 2008). In other studies, effects of seeding rate

on disease incidence were either very low (Turkington et al., 1991) or non-existent

(Nordin et al., 1992). In this study, seeding rate and fertilizer treatments were effective in

modifying canopy density. Overall, seeding and fertilizer treatments created significantly

different LAI values and plant counts among the four site-years, especially among high

and low seeding rate treatments. Canopy density treatments were effective in modifying

several microclimatic parameters as well. As expected, under denser canopies

temperature was decreased and relative humidity was increased significantly among 3 site

years. Soil temperatures were also decreased significantly under denser canopies. Leaf

wetness was significantly higher during one site-year. These sub canopy modifications

had no influence on airborne ascospore concentrations among the three density

treatments. Each treatment consisted of a 10 by 10 meter block which may not have been

large enough to accommodate spore dispersal distance. According to Williams and

Stelfox (1979), ascospores are capable of travelling up to 30 to 50 meters above the

surface and 150 meters from the apothecia source. Use of larger plots would allow for a

better capture of spores within each individual treatment. Also, because ascospores

originate from apothecia present at the soil surface, a denser canopy may not allow spores

to travel upwards beyond the foliage. Based on our study, we speculate that increased

92

relative humidity and leaf wetness and reduced temperatures influenced disease

development among denser canopies in Carman 2011; however these results were not

consistent throughout the other 3 site-years. As previously mentioned, denser canopies

may prevent ascospores from travelling up to the flowers for further colonization of the

plant. Additional study needs to be completed on the implication of dense canopies on

sclerotinia stem rot development.

Timely comparisons of ascospore concentrations in relation to below canopy

conditions yielded inconsistent results; however some trends were worth noting. Peaks in

relative humidity preceding peaks in ascospore concentrations demonstrate the

importance of humidity prior to ascospore release. Increased within canopy relative

humidity also led to higher leaf wetness values. Leaf wetness is important for the

deposition and colonization of ascospores on canola petals and further to the development

of disease within the entire plant (Bardin and Huang, 2001). High leaf wetness values

during and following spore deposition on canola may have led to increased disease

incidence among Carman plots during both years. Higher temperatures experienced in

Winnipeg may have also led to reductions in ascospore survival as temperature and

radiation influence survival rates (Canola Council of Canada, 2013).

Misting provided inconsistent results with respect to canopy density and disease

development in both Winnipeg and Carman fields in 2011; however misting did alter

microclimates slightly. Under misted plots, relative humidity and leaf wetness values

were increased, while soil and air temperature values were decreased slightly at both

Winnipeg and Carman locations. As a result of misting, microclimates were modified

93

mainly through the changes in macroclimate and not entirely due to changes in canopy

structure. In Carman, number of plants per square metre was higher in misted plots;

however leaf area index values remained lower indicating that although there were

additional plants, increased plant cover and densities were not achieved. In Winnipeg,

leaf area index was overall slightly higher and plant counts were lower in misted plots

indicating that individual plant densities were slightly higher, however fewer plants

appeared. Significant changes in canopy structure would have occurred if misting had

begun at the beginning of the growing season. Although spore concentrations and disease

incidence values were slightly higher among misted plots in Winnipeg, this did not occur

in Carman in 2011. Along with even inoculation of the plots, the Carman plot (2011) had

a previous history of disease mainly in the north portion of the field (personal

communication with Alvin Iverson, Ian N. Morrison Field Station, 2011). Since misting

occurred in the south portion, there may be a larger influence from the additional

inoculum source to the north creating increased ascospore concentrations and disease

incidences in that location of the field regardless of the absence of a misting system. Blad

et al. (1978) studied S. sclerotiorum in a semi-arid climate, where microclimates are

unfavourable for white mold disease in bean. A continuation of this work was completed

by Weiss et al. (1980). Both studies determined that, through the use of an irrigation

system, disease was most prominent under misted plots due to favorable microclimatic

conditions caused by the misting and by the advanced crop development which occurred

as a result of misting (Blad et al., 1978; Weiss et al., 1980). Temperatures in the air, on

leaves and soils remained cooler under misted plots and leaf wetness periods were longer.

The locations at which the fungus S. sclerotiorum was studied (eastern Colorado and

western Nebraska) have unfavourable conditions for disease with warm arid climates. In

94

this region, the days that were the hottest showed greater microclimatic changes through

the use of a misting system (Weiss et al., 1980). Use of a misting system in these

locations is more effective in substantially modifying microclimates and creating an

environment where disease can develop compared to Manitoba, which already has the

necessary conditions for disease development without the use of a misting system,

especially during years with adequate precipitation and cooler growing season

temperatures. Changes to the microenvironment may not be substantial enough to elicit

significantly more disease incidence in a prairie region such as in the province of

Manitoba.

Several confounding variables have made it difficult make accurate comparisons

among all four site-years. For instance, inoculation in 2012 occurred during both the fall

and spring prior to the growing season, whereas 2011 received inoculation in the spring.

Inoculums were also increased in 2012. Fall applied inoculum were able to overwinter in

the soil where they would germinate the following season compared to inoculum that

were conditioned outdoors in mesh bags and placed in the field during the spring. Both

experimental locations contained no history of disease and previously grown crops were

not susceptible to sclerotinia in 2012. This is similar for Winnipeg in 2011. Carman

(2011) contained history of disease in half the plot, therefore an increased level of

inoculum was present at that location which may be responsible for increased SSR

disease. Misting was provided to the crop in 2011 only, which may have influenced soil

moisture and overall relative humidity and temperature throughout the field making it

difficult to compare with 2012. As a result of not misting in 2012, two randomized

complete block designs were used in the analysis of natural non misted conditions

compared to only one randomized complete block design used in 2011. There was a

95

switch in the placement of microclimate stations and rotorods from 2011 to 2012.

Rotorods were placed at 2.5 meters from the edge of each plot in 2011 and in the middle

of each plot in 2012 to better reflect spores being captured mostly from within the plot

increasing distance to adjacent plots. Microclimate stations were in the middle of each

plot in 2011 and at 2.5 meters from the edge of each plot in 2012 to allow rotorod

placement in the center of each plot. Rotorods used in Winnipeg had retracting heads that

extended the polysterene rods only when the rods were spinning. Rotorods placed in

Carman had polysterene rods that were continually downward facing, which may be

responsible for increased spore concentrations found in Carman during both years. In

order to make comparisons among the treatments to adequately reflect their impacts on

microclimate and disease development, confounding effects need to be eliminated.

96

2.6 Conclusion

Comparing all four site-years, no correlation was observed between ascospore

concentrations and percent infection; however inoculum levels may have been effective

in increasing ascospore release. There appears to be an overall influence of weather on

ascospore concentrations and percent infection. Peaks in ascospore concentrations

occurred simultaneously between the two Manitoba locations separated by more than 50

km. Carman experienced high relative humidity and lower temperatures both above and

below canopy which led to increased infection compared to Winnipeg during both years

indicating that relative humidity is required for disease development. A precipitation

event that resulted in increased relative humidity preceding a decrease in relative

humidity and increase in temperature may have contributed to increased ascospore

releases, which concurs with previous research.

The misting system used to alter microclimates below the canopy in 2011 was not

effective in increasing ascospore release concentrations or percent infection at harvest.

Misting was effective in reducing within canopy temperature and increasing relative

humidity and leaf wetness. Ascospore concentration and percentage of infection were

higher among misted plots in Winnipeg, however this did not occur in Carman which is

likely due to the additional inoculum levels present in Carman.

Seeding rate and fertilizer treatments were effective in significantly modifying

LAI and plant counts among high versus low density plots. Several microclimatic factors

also proved to be significantly different between high and low treatments among all site-

years, including air temperature, soil temperature and relative humidity. These

97

modifications had no influence on airborne ascospore concentrations. Denser canopies

showed significantly higher infection values in Carman during 2011; however no

significant differences were seen among the other 3-site years.

This study concludes that modifying canopy density and providing additional

moisture through misting does alter microclimate, however not enough to significantly

affect disease development. The overall seasonal weather will have an impact on disease

development; however inoculum concentrations seem to have a greater impact on disease

under favourable conditions with adequate moisture and optimum temperatures.

98

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Blad, B. L., Steadman, J. R., and Weiss, A. 1978. Canopy structure and irrigation

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potential on sclerotia germination and apothecial production of Sclerotinia sclerotiorum.

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canopy microclimate and white mold disease (sclerotinia sclerotiorum) development in

dry edible beans as influenced by canopy structure and irrigation. Agricultural

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3. INFLUENCE OF NON-HOST CROPS IN NEIGHBOURING FIELDS ON SCLEROTINIA STEM ROT DISEASE

3.1 Abstract

Crop rotation is a widely used management strategy influencing canola yields and

reducing pressures from unwanted pests and diseases including Sclerotinia sclerotiorum.

Canola fields free of disease history may still be at risk of being infected due to the ability

of sclerotinia ascospores to travel from neighboring fields, including fields containing

non-host crops. This study compares ascospore release under both wheat and canola

canopies and analyzes the influence of weather and microclimate on airborne ascospore

concentrations in a wheat field. Gradients were created alongside the wheat field to

monitor spore dispersal distance as well as the influence of wind direction on ascospore

dispersal. This study demonstrated that sclerotia germinate and ascospores are released

from apothecia within a wheat canopy at similar and sometimes greater rates than release

under a canola crop. Further, ascospores are capable of traveling at distances of greater

than 100 m into neighboring fields. Although crop rotation is an important disease

management strategy, its effectiveness in reducing inoculum levels is unclear. Disease

forecasting also needs to incorporate information on neighboring fields and their history

of disease.

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3.2 Introduction

Sclerotinia stem rot (SSR) disease is a serious disease affecting over 400 host

crops worldwide caused by the fungus Sclerotinia sclerotiorum (Lib.) de Bary. In canola,

ascospores are the primary source of inoculum of Sclerotinia sclerotiorum (Jamaux et al.,

1995) and create most primary infections occurring in the field (Abawi and Grogan,

1979). Ascospore release and dispersal is important due to their impact on SSR disease

intensity in canola (Qandah, 2008). During the growing season, ascospores are produced

carpogenically from sclerotia, which are overwintering fungal structures resting at or near

the soil surface and capable of surviving in the soil for prolonged periods of time.

Ascospores are forcibly released into the surrounding atmosphere by asci contained

within apothecia which develop from sclerotia (Harthill and Underhill, 1976). SSR is

considered to be a monocyclic disease containing only a single infection cycle. During

this cycle, several cohorts of airborne ascospores released into the air land on petals

which serve as a source of nutrition and the infected petals subsequently fall on nearby

stems and leaves and infect and potentially kill entire plants. Timing of ascospore release

is critical for disease occurrence; release must occur during the canola flowering stage.

Studies to date have focused mainly on the environmental factors associated with

the release of ascospores within SSR sclerotinia stem rot susceptible crops. Light,

temperature and relative humidity have been studied extensively by researchers.

Harthill‟s (1980) study indicating that light was involved in the process was later

overturned by Clarkson et al (2003) who proved that spores were released under both

light and dark conditions. Significant impacts of relative humidity and temperature on

105

release of ascospores were reported by Qandah and Mendoza (2011). Their study

concluded that ascospore release occurs only once daily in a single event and lasts no

longer than six hours with the release in wet years occurring only during the day and only

during the night in dry years (Qandah and Mendoza, 2011). Ascospores were generally

released in periods of increasing air temperatures, and decreasing relative humidity

following prolonged periods of high relative humidity (Qandah and Mendoza, 2011).

Previous findings also conclude that ascospore release occurs with decreasing relative

humidity and increasing temperatures and wind speeds (McCartney and Lacey, 1991).

Although these environmental factors affecting ascospore release have been documented,

additional emphasis needs to focus on seasonal release and dispersal of spores and effects

of additional factors including wind speed and direction.

The few studies focusing on S. sclerotiorum ascospore dispersal have found that

disease results mainly from inoculum produced within the field (Boland and Hall, 1988;

Twengstrom et al., 1998). In contrast, a study specifically focusing on Sclerotinia

ascospore dispersal in canola was completed by Qandah and Mendoza (2012) in which

dishes containing a semi-selective medium were placed throughout a canola field at

several distances from a known source of inoculum. Disease and ascospore

concentrations declined with distance from the source of inoculum (Qandah and

Mendoza, 2012). No ascospores were dispersed in dry years with little precipitation and

low relative humidity values (Qandah and Mendoza, 2012). Similar studies have been

completed on other fungal pathogens with similar characteristics. Guo and Fernando

(2005) conducted a study in Manitoba on ascospore dispersal by Leptosphaeria maculans

in relation to environmental conditions. Burkard and rotorod spore trapping devices were

106

placed throughout two fields to capture spores. Hourly and daily ascospore concentrations

were recorded from the devices and plotted against weather values. The effect of wind

direction on ascospore dispersal was analyzed by monitoring ascospore concentrations at

several distances from the inoculated plots. Fungal ascospore concentrations of L.

maculans peaked during and following rainfall events when relative humidity was

elevated and temperature decreased. Ascospore concentrations also decreased with

distance from the inoculated source and wind direction had a significant effect on

ascospore dispersal where increased concentrations were found downward from the wind

source (Guo and Fernando, 2005). Quantification of ascospore concentrations in relation

to several weather factors including wind direction was completed successfully for L.

maculans and a similar analysis needs to be completed for S. sclerotiorum.

Previous studies have characterized only a few environmental conditions

necessary for release and dispersal of ascospores in crops susceptible to S. sclerotiorum.

A better understanding of disease development under a crop not susceptible to S.

sclerotiorum is required to determine the effectiveness of disease management of

sclerotinia by crop rotations using non-host crops. An evaluation of wind speed and

direction on dispersal of ascospores and their potential to infect neighboring fields is also

important. Little is known on the influence of non-host crops and sclerotinia disease. The

objective of this study was to determine the influence of a neighbouring non-host wheat

crop on sclerotinia stem rot disease in canola, the impact of microclimatic factors within

the canopy, as well as wind speed, wind direction and ascospore dispersal above and

adjacent to the canopy.

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3.3 Methods

3.3.1 Study Sites and Field Layout

Two field sites were used in 2011 and 2012 to study the effects of sclerotia-

inoculated wheat fields on ascospore production and dispersal. Fields were located in

Winnipeg and Carman. In 2011, the field used in Winnipeg contained no previous history

of disease, whereas the field in Carman contained a history of disease in the previous

years. Neither field site in 2012 had a previous history of disease and had been seeded

with non-host crops in prior years.

In Winnipeg (2011), the south portion of the plot was seeded with 3 density

treatments of canola over 18 plots using a randomized complete block design as

explained in chapter 2. The remaining field portion to the north, had „Kane‟ wheat seeded

in a strip running from north to south (10m wide by 50m long), with 10m of bare soil to

the east and west of the wheat strip. In 2012, the north portion of the field was seeded

with canola, while the remaining south field portion had AC Barrie wheat running from

north to south with bare soil to the east and west of the strip. Similarly, in 2011 and 2012

Carman was seeded with a canola field portion containing 3 treatments over 18 plots. The

remaining portion of the field to the left contained „Kane‟ wheat (10m by 50m) with 10m

of bare soil on each side. The canola field portion ran from west to east in Carman in

2011, with the wheat strip to the north. In 2012, the canola portion ran from east to west

with the wheat strip located to the south (A.3).

108

3.3.2 Field Preparation

Wheat (Kane in 2011 and AC Barrie in 2012) and canola seeding in Winnipeg and

Carman occurred on May 26th

and June 8th

in 2011 and on June 6th

and May 23rd

in 2012

respectively. Carman received tillage in 2011, Winnipeg did not. Wheat was seeded at

130kg/ha and ammonium sulphate (30 kg/ha), MAP (30 kg/ha) and urea (200kg/ha)

fertilizers were used for all site years. Field preparation in 2012 was similar to 2011,

however pre-emergent herbicide (Edge) was applied and incorporated into the soil on the

canola portions of both fields in the fall of 2011 for weed control in 2012. The field in

Carman was tilled prior to the 2012 growing season and the field in Winnipeg was not

tilled. Post-emergent herbicides (Axial and Infinity) were sprayed to further eliminate

grass and broadleaf weeds in Carman.

Inoculum in the form of conditioned sclerotia was prepared by placing the

sclerotia within mesh bags outside in snow in January and left covered throughout the

winter. Mesh bags were then moved to a refrigerator upon snowmelt to avoid

germination. Both the Carman and Winnipeg sites were inoculated with conditioned

sclerotia after seeding in 2011. The wheat strip received 360 grams of equally dispersed

sclerotia, at the same rate as sclerotia applied to the canola. Fall field preparation in 2012

included the inoculation of 400g sclerotia onto each of the Winnipeg (November 2nd

,

2011) and Carman (October 26th

, 2011) field sites. An additional 240g of sclerotia was

applied to the wheat and canola strips in the spring on June 5th (Winnipeg) and June 14

th

(Carman), respectively.

109

3.3.3 Microclimate Monitoring

HOBO microclimate stations were used to monitor microclimate during the

growing seasons (2011 and 2012) in both wheat and canola plots. Measures for

microclimate included air temperature, relative humidity, leaf wetness, surface soil

temperature and surface soil moisture. Two microclimate stations were placed within the

wheat strips at each location; one in each of the misted and non-misted wheat portions in

2011 and one in each of the replicated field layouts in 2012. One microclimate station

was also placed within each of the 18 canola plots. Placement of the stations in Winnipeg

and Carman occurred on June 6th

and June 17th

, respectively, in 2011. In 2012, stations

were placed in the field on June 26th and June 13

th in Winnipeg and Carman, respectively.

Soil moisture and soil temperature probes were placed in the soil vertically using a

trenching shovel to dig holes; the surrounding area was replaced with soil. Air

temperature probes and relative humidity sensors were placed at 10 cm above the soil

surface.

Leaf wetness sensors and relative humidity and temperature sensors within the

solar radiation shield were adjusted throughout both growing seasons. Leaf wetness,

temperature and relative humidity sensors were placed at 12 cm in Winnipeg and Carman

on June 28th

and July 17th respectively in 2011 and on June 26

th and June 13

th respectively

in 2012. In 2011, leaf wetness, temperature and relative humidity sensors were raised to

24 cm on July 7th

(Winnipeg) and July 21st (Carman). In 2012, sensors were placed at 24

cm on July 4th (Winnipeg) and June 21st (Carman).

110

3.3.4 Ascospore Dispersal

Rotorods were installed throughout each field to monitor the dispersal of spores

from both canola and wheat field portions. The rotorod collected spores on 2 polystyrene

collector rods that spun at 2400 RPM for 1 minute out of every 10 minutes. Rotorod

monitoring in 2011 began on June 30th (Winnipeg) and July 11

th (Carman). In 2012,

rotorods were monitored beginning on June 27th

in Carman and July 7th

in Winnipeg.

Within both fields, one rotorod was placed in each of the canola plots, and 4 rotorods

were placed within the wheat strip. In 2011, two were placed in each of the misted and

non-misted areas, whereas in 2012 two rotorods were placed in each replicate of the

design. Eight rotorods were also placed in the bare soil on each side of the wheat strip.

Four rotorods were placed at 3 meters and four at 7 meters from the inoculated wheat

strip. The purpose for which spore samplers were placed on the bare soil field portions

were to monitor the distance and direction that spores were dispersed on either side of the

wheat and to understand disease gradients. All rotorod samplers placed in Winnipeg

contained retracting heads, while samplers in Carman contained fixed heads. Rods were

replaced daily for further analysis in the lab.

Ascospores of S. sclerotiorum on each collector rod were counted in the lab using

a microscope at 400x magnification as described in Chapter 2. The total number of

ascospores per rod was calculated based on the counts obtained in each lens view as

described in Chapter 2.

The values obtained for each of the two rods were then averaged to get a mean

value of ascospores per metre cubed per rotorod per day.

111

Occasionally rods could not be counted or contained no spores for several reasons.

Rods that could not be counted properly had lots of dirt on them, large sections of the rod

were covered by a bug or large sections of the rod contained no spores. Rods containing

either no spores or dirt on them were generally placed in the rotorod backwards and also

could not be counted. Some rods were also missing meaning that they may have been

dropped in the field and not found. Where only one of the two rods from a rotorod was

counted, the value from that rod alone was counted as the mean daily ascospore

concentration. Where none of the two rods could be counted, gaps in the data were

created.

3.3.5 Misting

During the 2011 growing season, half of the wheat strips and half of the canola

field portions in each field were misted to ensure continual moisture for disease

development and to provide a range of microclimatic conditions using the system

described in Chapter 2. The remaining halves were not misted to represent standard

weather conditions. In Winnipeg, plots 10 through 18 and the south half of the wheat strip

were misted, and in Carman, plots 1 through 9 and the west side of the wheat strip were

misted. Misting in 2011 began at the 20% bloom stage on July 5th

and July 15th

in

Winnipeg and Carman, respectively. Misting was terminated ten days after the final

canola petal had fallen on July 31st (Winnipeg) and August 10

th (Carman). Misting began

daily at 4 pm and was left on until the 1,200 gallons (4,542 L) emptied. Approximately

26,250 gallons (99,367 L) of water was irrigated onto the misted field portions in total.

112

Neither of the Winnipeg or Carman plots were misted in 2012 due to technical

issues and availability of generators and pumping systems. This created two sets of

randomized complete block designs in each of the Winnipeg and Carman canola fields.

3.3.6 Growing Season

Length of the growing season from seeding to harvest, the period before flowering

from 4th

leaf to flowering, flowering stage and the ascospore sampling period dates are

outlined below in Table 3.1.

Table 3.1 Start and end dates for specified time periods during the growing season.

Time Period Carman 2011 Winnipeg 2011 Carman 2012 Winnipeg 2012

Growing season June 8-Aug 22 May 26-Aug 5 May 23-Aug 7 June 6-Aug 13

Before flowering June 29-July 14 June 15-July 4 June 16-July 11 June 27-July 10

During flowering July 14-Aug 6 July 5-July 21 July 1-July 22 July 11-Aug 1

Sampling period July 11-Aug 17 July 4-July 29 June 27-July 25 July 7-Aug 6

113

3.3.7 Data Analysis

Recorded weather and microclimate parameters for wheat were summarized for

the sampling period and during several growing season intervals for comparison among

field locations and between years. Daily microclimate values were plotted with daily

mean ascospore concentrations within the wheat for comparison of trends. Comparisons

of average daily mean ascospore concentrations and several microclimate parameters

including air temperature, soil temperature, relative humidity and leaf wetness were made

between misted and non-misted field portions in 2011.

An analysis was completed on wind direction relative to spore dispersal through a

series of comparisons of daily mean ascospore concentrations in the direction of the wind.

Comparisons of wind direction on ascospore dispersal within the wheat strips were made

by comparing ascospore concentrations over days with similar dominant wind directions

in Carman in 2011 and 2012 and in Winnipeg in 2012. The wheat strip located in Carman

2011 ran from north to south; daily ascospore concentrations within the wheat strip were

compared with wind direction. Ascospore concentrations obtained from both rotorods to

the north were averaged to obtain a mean daily ascospore concentration in the north

direction and the same was done for the ascospore concentrations within the south of the

wheat strip. Some analysis also focuses on distance of dispersal using the data available

through the project design.

114

3.4 Results

3.4.1 Weather and Microclimate Impacts on Ascospore Release in the Wheat Plots

During the rotorod sampling period, daily mean air temperatures were higher

overall in 2012 compared to 2011, however growing season temperatures were similar

among siteyears (Table 3.2). Daily mean air temperatures in Winnipeg and Carman were

comparable during the growing season; however Winnipeg temperatures tended to be

slightly higher than Carman temperatures during the sampling period in 2011. Higher

maximum temperatures were attained in Winnipeg compared to Carman during each year.

Mean relative humidity was higher in Carman in 2011 and 2012 during both time periods

with slightly higher percentages occurring in 2011. Total precipitation during the growing

seasons was higher in 2012. In 2011, Winnipeg received more precipitation than Carman

overall, however in 2012 Carman received more precipitation during the growing season.

During the rotorod sampling period, Winnipeg received more in both years. Data for

dominant wind direction in Winnipeg in 2011 are not available.

Below canopy daily mean air temperatures within the wheat strip were higher in

Carman compared to Winnipeg throughout most of the season in 2011, whereas daily mean

air temperatures were higher in Winnipeg in 2012 overall (Table 3.3). Carman showed

higher maximum temperatures in 2011, and Winnipeg showed higher maximum

temperatures overall in 2012. Relative humidity under the wheat canopy was higher in

Carman during both years, with increasing relative humidity during the flowering stages

during most site-years. Overall, relative humidity was higher during the growing season at

both locations in 2011. Leaf wetness was consistently higher in Carman during both

115

seasons; with higher values for average leaf wetness during the flowering stages in all site-

years.

Average daily values for relative humidity and leaf wetness followed a similar trend

throughout the spore sampling period during all site years (Figures 3.1 and 3.2). Air

temperature and soil temperature were also similar throughout each period. There appears

to be no correlation between average daily temperature and relative humidity values. In

most cases, peaks in ascospore concentrations tended to occur following increases in within

canopy relative humidity with peaks in spore concentrations occurring 1-2 days after peaks

in relative humidity values. Canopy temperature and ascospore concentrations did not show

any temporal correlation. Average mean daily ascospore concentrations in Winnipeg and

Carman peaked during similar periods in 2011 and 2012. In 2011, major peaks occurred

around July 24th

and July 27th

at both sites; major peaks occur around July 17th

and July

20th in 2012.

116

Table 3.2 Rotorod sampling period and growing season weather parameters above the

canopy for Winnipeg and Carman in 2011 and 2012. Maximum and minimum

temperatures are indicated in brackets beside mean temperature values.

Daily mean air temperature (˚C)

Mean relative humidity (%)

Total precipitation (mm) Dominant wind direction

Winnipeg Carman Winnipeg Carman Winnipeg Carman Winnipeg Carman

Time Period 2011

Sampling Periody 22 (34, 10) 20 (33, 9) 62 73 31 24 NA

z West

Growing Seasonx 20 (34, 0) 20 (33, 5) 65 72 162 135 - -

2012

Sampling Periody 23 (35, 10) 23 (34, 12) 62 70 53 35 South West

Growing Seasonx 20 (35, 1) 20 (34, 3) 64 71 129 208 - -

z Data is unavailable ySampling Period - Wpg2011 (July 4 – July 29), Car2011 (July 11 – Aug 17), Wpg2012 (July 7 – Aug 6), Car2012 (June 27 – July 25) xGrowing Season – Wpg2011 (May 26- Aug 5), Car2011 (June 8 – Aug 22), Wpg2012 (June 6 – Aug 13), Car2012

(May 23 – Aug 7)

Table 3.3 Microclimate parameters within the wheat strips in Winnipeg and Carman in

2011 and 2012 during the rotorod sampling period, from 4th leaf to flowering stage

and during the flowering stage for canola. Calculated values are obtained from the

average of two microclimate stations placed within the wheat strips.

Daily mean air temperature

(˚C) Average relative

humidity (%)

Average soil

temperature (˚C) Average leaf wetness

Winnipeg Carman Winnipeg Carman Winnipeg Carman Winnipeg Carman

Time Period 2011

Sampling Periody 20 (22, 17) 20z (25, 15) 80 88

z 21 20z 37 56

z

4th Leaf to Floweringx 19 (22, 17) 21 (25, 17) 80 81 20 21 20 24

Flowering Stagew 20 (22, 17) 21z (25, 15) 80 90

z 21 21z 38 58

z

2012

Sampling Periody 22 (26, 16) 23 (26, 17) 78 82 22 22 36 45

4th Leaf to Floweringx 23 (25, 21) 18 (23, 13) 73 80 23 20 30 39

Flowering Stagew 23 (26, 17) 23 (26, 17) 77 85 22 22 32 47

z Missing values from one of the rotorods from Jul 28 to Aug 12. Where missing values are present, only one

microclimate station represents the average for the wheat strip y Sampling Period - Wpg2011 (July 4 – July 29), Car2011 (July 11 – Aug 17), Wpg2012 (July 7 – Aug 6), Car2012

(June 27 – July 25) x 4th Leaf to Flowering – Wpg2011 (June15 – July4), Car2011 (June29 – July14), Wpg2012 (June27 – July10),

Car2012 (June 16 – July 11) w Flowering Stage - Wpg2011 (July5 – July21), Car2011 (July14 – Aug6), Wpg2012 (July11 – Aug 1), Car2012

(July1 – July22)

117


(black line), and soil temperature in ˚C (grey line) and C) leaf wetness (black line)

and relative humidity in percentage (grey line) to A) daily mean ascospore

concentrations (ascospore/m3) in I) Carman and II) Winnipeg in 2011. Red lines

indicate the beginning and the end of the flowering period.

118


(black line), and soil temperature in ˚C (grey line) and C) leaf wetness (black line)

and relative humidity in percentage (grey line) to A) daily mean ascospore

concentrations (ascospore/m3) in I) Carman and II) Winnipeg in 2012. Red lines

indicate the beginning and the end of the flowering period.

119

A comparison was made between misted and non-misted wheat field portions in

Winnipeg and Carman in 2011 using microclimate and daily mean ascospore

concentrations (Table 3.4). Calculated values were obtained for the sampling period

which began just prior to flowering to several days after flowering at both locations.

Compared to non-misted field portions, misted portions contained higher average daily

mean ascospore concentrations in Carman, however concentrations were lower among

misted plots in Winnipeg. Daily mean air temperatures, relative humidity values and daily

mean soil temperatures were very similar between misted and non-misted wheat field

portions in both Winnipeg and Carman. Leaf wetness was higher among misted plots at

both locations in both wheat and canola.

Table 3.4 Measured values for ascospore concentrations and microclimate parameters in

Winnipeg and Carman in 2011 for misted and non-misted wheat field portions

during the sampling period.

Location Sampling Period Treatment

Average daily mean spore

concentration (ascospores/m

3)

Daily mean air

temperature (°C)

Relative

humidity (%)

Leaf

wetness in wheat

Leaf

wetness in canola

Daily mean soil

temperature (°C)

Carman Jul 11 – Aug 14

Misted 684y (29 days) 20

z 88

z 59

z 62 20

z

Non-misted 653

y (29 days) 20

z 89

z 49

z 47 20

z

Winnipeg Jul 4 – Jul 29

Misted 646x (20 days) 20 80 40 45 21

Non-misted 823

x (20 days) 20 80 35 30 21

zmissing values during sampling period; only values for days available were used (Jul 11-27:Aug 13-14)

yvalues obtained from 1 rotorod in each treatment xvalues obtained from 2 rotorods in each treatment

120

3.4.2 Impact of Wind on Ascospore Dispersal from the Wheat Plots

In Carman in 2011 and 2012 during the sampling period, prevailing winds were

predominantly westerly. In Winnipeg in 2012, winds were predominantly southerly; wind

direction was not monitored throughout the entire sampling period in 2011 (Figure 3.3).

Values for average daily mean ascospore concentrations over the sampling period were

generally higher within the wheat strips compared to the bare soil field portions, especially

in Carman. There were no other noticeable trends occurring as a result of predominant wind

directions during the spore sampling period.

121

Figure 3.3 Design of bare and wheat field portions for A) Carman 2011 B) Winnipeg

2011 C) Carman 2012 and D) Winnipeg 2012. Circles represent rotorod locations

with average daily mean ascospore concentrations within them for the sampling

periods at each field at both locations. Black arrows in the bottom right corners

represent dominant wind directions during the sampling periods. Rotorods were

placed at 3 and 7 metres from inoculated wheat strips in bare soil. Image is not to

scale.

122

In Carman (2011), days with northerly predominant wind directions (including

winds blowing towards the south, south-west and south east) or southerly predominant

winds (including winds blowing towards the north, north-east and north-west) showed

higher daily mean ascospore concentrations in the downwind direction during 50% of the

days. In 2012, daily mean ascospore concentrations in the upwind direction within the

wheat strip were higher within 64% and 58% of the plots in both Carman (running from

east to west) and Winnipeg (running from north to south) respectively. The Winnipeg plot

in 2012 contained a canola field portion directly to the north which may have affected

spore concentrations in the north portion of the wheat strip.

Ascospore concentrations within the bare soil areas on either side of the wheat

strip in Winnipeg, 2012 were also compared based on easterly (including winds blowing

from the north-east, south-east and east) and westerly (including winds blowing from the

north-west, south-west and west) winds. An average of all ascospore concentrations on

the east and west sides were compared based on wind directions. Spore concentrations

downwind from the prevailing winds were higher for approximately 71% of the days with

easterly or westerly winds.

During certain days in Carman, prevailing winds were from the direction where

canola plots were not situated adjacent to the bare soil portion. In 2011 and 2012, these

winds were from the west and south respectively. Spore concentrations downward from

the prevailing winds were higher for 50% and 40% of the days in 2011 and 2012

respectively.

123

Distance of dispersal was analyzed by comparing daily mean ascospore

concentrations averaged over the sampling period at 3 meters and 7 meters from the

inoculated wheat field. In Winnipeg in 2011 and 2012, there is a slight decreasing trend in

average daily mean ascospore concentration with increasing distance from the inoculated

source (Figure 3.3). A slight decrease in average daily mean ascospore concentration was

also apparent in Carman in 2011; however an increasing trend was apparent in 2012.

Ascospore concentrations tend to follow a negative exponential relationship with distance

away from an inoculated source (Qandah and Mendoza, 2012). This form of model was

utilized to analyze the distance relationship in Winnipeg in both years. The Winnipeg

plot in 2011 showed a slightly steeper slope than that for 2012 (Table 3.5). A 50 percent

percent reduction in daily mean spore concentrations from the inoculum source averaged

over the sampling period would occur at approximately 61 and 62 meters in 2011 and

2012, respectively. A 75 percent reduction would occur at approximately 119 and 122

meters in 2011 and 2012, respectively.

Table 3.5 Parameters obtained by an exponential model applied to mean spore concentrations at

3 meters and 7 meters from an inoculum source in Winnipeg. 50% reduction and 75%

reduction indicate the distance at which a 50 percent and 75 percent reduction in average mean spore concentration would occur.

Site Year slope y-intercept 50% reduction (m) 75% reduction (m)

Winnipeg 2011z -0.01186 693.23 61.4 119.9

Winnipeg 2012 -0.01168 680.44 62.3 121.6 z Only 1 value for average daily mean ascospore concentration at 7 m was used

124

3.43 Dispersal Comparisons among Wheat and Canola Plots

A comparison of average daily mean ascospore concentrations over the sampling

period at 3 meters from the inoculated wheat and 3 meters from the inoculated canola

fields within the bare soil field portion was done. Consistently higher concentrations

were realized for the rotorods within the bare soil portions closer to the canola plots

compared to the wheat plot in Carman during both 2011 and 2012 (figure 3.3).

In Carman canola plots (2011 and 2012), average daily mean ascospore

concentrations were lowest at 10 to 20 metres from the wheat inoculum source (Table

3.6). Concentrations increased with distance from the wheat inoculum source. There were

no consistent trends relating disease incidence to distance from the wheat inoculum

source. In 2011, disease incidence was highest among plots at 20 to 30 metres and in

Winnipeg disease incidence was highest within plots at 30 to 40 metres.

Table 3.6 Average daily mean ascospore concentrations (ascospores/m3) and disease

incidence averages within canola plots at 30-40, 20-30 and 10-20 metres from

inoculated wheat source in 2011 and 2012 Carman plots.

Ascospore Concentration Disease Incidence

Distance from wheat source 2011 2012 2011 2012 30-40 metres 655.3 1894.41 18.6042 17.9945 20-30 metres 621.7 1821.87 30.1623 13.5664 10-20 metres 577.21 1821.32 15.9979 15.4809

125

In 2011 at both sites, average daily mean ascospore concentrations were higher

among wheat plots in all plots, misted plots and non-misted plots, whereas in 2012

concentrations were higher within canola (Table 3.7). Mean daily ascospore

concentrations under wheat and canola canopies followed similar trends (Figure 3.4).

Percent infection at harvest was lower overall in 2011 compared to 2012 with higher

values occurring in Carman during both years.

Table 3.7 Whole plot means for average daily mean ascsopore concentrations in wheat

and canola plots and percent infection obtained at harvest in canola plots at

Winnipeg and Carman in 2011 and 2012. Ascospore concentration data used was

taken only for days in which all values were present in both wheat and canola

during a specified site year over the sampling period.

Year Site Ascospore

Sampling Period Description

Ascospore Concentrations (ascospores/m3)

in Canola

Percenta Infection

(%) in Canola

Ascospore Concentrations (ascospores/m3)

in Wheat

2011

Winnipeg July 6- July 26 All 576 5.8 623 (12 days) Non-Misted 571 6.0 609 Misted 581 5.5 637

Carman July 12- August

14 All

655 22

790 (14 days) Non-Misted 714 23 789 Misted 596 21 791

2012

Winnipeg July 8- August 6

(23 days) All (Non-Misted) 769 3 and 9z 651

Carman June 29- July 23

(13 days)

All (Non-Misted) 2008 16 1491

z Value was obtained post-harvest

126

Figure 3.4 Average daily mean ascospore concentrations in wheat (black line) and canola

(grey line) throughout the sampling period in A) Carman 2011, B) Winnipeg 2011,

C) Carman 2012 and D) Winnipeg 2012.

A

B

C

D

127

3.5 Discussion

To the best of our knowledge, this is the first study to investigate the development

and spread of S. sclerotiorum in a non-host crop. This study has provided evidence

that ascospore production is possible under a non-host wheat crop and ascospore release

concentrations and timing of peak levels are similar to those under a canola canopy.

Ascospores were captured at a distance of 7 meters and it has been reported that travel

distance can exceed 60 meters (Qandah and Mendoza, 2012). Climatic conditions

necessary for ascospore release and infection were increased relative humidity and leaf

wetness and reduced temperature. Wind direction also had a slight impact on ascospore

dispersal away from the wheat source. Misting was not effective in increasing ascospore

concentrations based on this study.

Weather and microclimate are important factors for both crop and disease

development. According to Turkington et al. (2011), risk of stem rot is increased under

specific environmental conditions. During the period prior to flowering, adequate rain and

moderate temperatures are required to allow for soil surfaces to remain wet for the

majority of the day and enhance sclerotial germination (Turkington et al., 2011). These

conditions are also necessary under a wheat canopy for successful germination of

sclerotia and release of ascospores into the surrounding air. In 2011, seeding occurred

later throughout most of Manitoba due to the saturated top soils, heavy snow melt and

precipitation occurring throughout May and June. The rest of the summer remained fairly

dry and hot. In 2012, warm dry weather in April was followed by a prolonged cool period

at the beginning of May. Warm dry conditions ensued throughout the summer. Overall

128

temperatures were slightly warmer and relative humidity percentages were lower in 2012

which reduced the canola flowering period, reducing seed set and size. Compared to

Carman, Winnipeg experienced higher maximum temperatures and lower percent relative

humidity during both years due to the urban location of the Winnipeg plots. Below

canopy measures for microclimate indicated that soil and air temperatures were higher

overall in 2012 and relative humidity values were lower which is expected due to the

influence of weather during both years.

The use of a misting system to promote disease development was successful in

altering microclimates, however, it did not influence percent infection in this study. Due

to the operation of a misting system during canola flowering, relative humidity and leaf

wetness were much higher in wheat during the canola flowering period compared to the

leaf stages prior to flowering in Carman and Winnipeg in 2011. Relative humidity and

leaf wetness were also slightly higher in Winnipeg and Carman in 2012 during the

flowering period compared to the leaf stages, however a misting system was not used

therefore the increases were not substantial. Moisture is required to increase relative

humidity within the wheat canopy during sclerotial germination and spore release;

however relative humidity was not higher overall under misted wheat field portions.

Ascospore production is therefore not likely increased as a result of misting. In 2011, leaf

wetness was the only microclimatic parameter that showed significantly higher values

under misted wheat field portions. Similarly in canola, misted plots contained higher

values for leaf wetness. Leaf wetness in a canola crop is important for the deposition and

colonization of spores, providing an availability of water for pathogens on both flower

and leaf surfaces (Huber and Gillespie, 1992). Misted plots within the canola canopy with

129

consistently higher leaf wetness values could have favoured deposition and development

of sclerotinia ascospores into disease epidemics throughout the crop. Greater S.

sclerotiorum infections would occur where moisture is provided to both host and non-host

crops throughout the entire growing season to allow for enhanced germination, spore

release, spore deposition and infection.

During the flowering period, predictions of major ascospore release periods in

adjacent crops are critical for the determination of necessary fungicide applications within

an adjacent susceptible crop. Ascospore release from apothecia within a wheat field is

synchronous with release occurring in a canola field due to the influence of weather and

microclimate within the canopies. In 2011, major peaks in ascospore concentrations

occurred during similar dates in Winnipeg and Carman which is affected by the overall

weather conditions throughout Manitoba. These conditions are still not completely

understood. In 2012, major peaks also occurred during similar dates in both Winnipeg

and Carman. In 2011, both Winnipeg and Carman experienced major peaks in ascospore

concentrations towards the middle (50 to 60% in Carman) to end (70% and after

flowering in Winnipeg) of the canola flowering stages. In 2012, major peaks in ascospore

concentrations in wheat occurred mainly around 30% flowering and with larger peaks at

80 to 90% flowering which would likely have influenced neighbouring canola fields both

early in the flowering period and near the end of flowering. Among all microclimatic

variables, relative humidity had the greatest impact on ascospore release within wheat

canopies. Major peaks in ascospore concentrations mainly preceded prolonged periods of

high relative humidity followed by a sudden decrease in relative humidity which agrees

with the findings suggested by Qandah and Mendoza (2011). Within susceptible crops

130

and in adjacent crops with a history of disease, identification of major peaks in ascospore

concentrations occurring early in the growing season is important for disease prevention.

Recommended fungicide application time in Manitoba is at 20 to 30% bloom (Manitoba

Agriculture, 2013), however disease protection is increased when fungicides are applied

when apothecia appears in the crop (Mwiindilila and Hall, 1989). The identification

process can be facilitated through weather predictions early in the flowering period by

assessing the likelihood for precipitation and conditions of increased relative humidity for

prolonged periods.

According to Qandah (2008), sclerotinia stem rot incidence is closely associated

with airborne ascospore concentrations. This can be applicable to disease incidences

observed in canola fields as a result of neighbouring infested fields containing non-host

crops. Apothecia are capable of producing spores within non-host crops and releasing

them into the surrounding atmosphere as confirmed by this study (by the atmospheric

spore counts obtained above wheat canopies), which were consistently higher than the

counts obtained above the adjacent bare soil field portions that did not contain inoculum.

Twengstrom et al. (1998) suggested that even where external sources of inoculum exist,

inoculum originating within the canola field has the greatest influence on disease

occurrence. The local inoculum source within each canola plot proved to be a bigger

factor contributing to disease incidence and ascospore dispersal within the canola than the

nearby wheat canopy in proximity as shown in this study. Adjacent field borne inoculum

still however does influence neighbouring fields as established in studies where ascospore

dispersal gradients were created. Qandah and Mendoza (2012) were able to collect S.

sclerotiorum ascospores below a canola canopy at greater than 60 m from an inoculated

131

source while observing infected plants at 63 meters from the source. Similarly, in a study

conducted by Guo and Fernando (2005), Leoptosphaeria maculans ascospore

concentrations were highest within 10 to 25 m from the source of inoculum. The Canola

Council of Canada (2014) recommends field separation of 50 to 100 meters from blackleg

infested fields planted to canola the previous year. These findings were further validated

within this study since S. sclerotiorum ascospores were capable of travelling at least 7

meters along the gradient used to monitor spore dispersal above the canopy. Ascospore

dispersal from a source of inoculum tends to follow a negative exponential gradient

(Qandah and Mendoza, 2012). The steepness of the gradient can be explained by spore

size, nature of the spores dispersal mechanism, sampling method and influence of weather

(Qandah and Mendoza, 2012). Using a negative exponential model used by Qandah and

Mendoza (2012) and data from 2 sampling locations in the bare soil at 3 and 7 metres

from the wheat inoculum source, spore dispersal distances were calculated in Carman.

Reduction in spore concentrations by 50 percent would occur at approximately 61 to 62

meters from the inoculum source using the mean values obtained for slope and y-

intercept. A 75% reduction would occur at 120-122 meters. This calculation however

does not take into consideration the additional contribution from the canola fields located

adjacent to the wheat, which also likely impacted spore concentration within bare soil

from long distances. Qandah and Mendoza (2012) found that precipitation influences the

steepness of ascospore dispersal gradients where gradients become shallower with

increasing precipitation. In Winnipeg, the slightly steeper slope in 2011 can be partially

explained by the reduced precipitation occurring during the growing season and sampling

period compared to 2012. The positive gradient occurring in Carman in 2012 may be

explained by strong prevailing winds increasing spore concentrations in the far spore

132

sampler to the south-east, or by potential presence of inoculum in adjacent fields.

Dispersal distances can be determined through the use of prediction models using

precipitation as a contributing factor. Growers should be aware of the history of disease

and inoculum levels in fields nearby. Fungicide applications may be required on

susceptible crops located adjacent to fields with disease history.

Climate is shown to be a major contributor to disease development as established

by ascospore concentrations and disease incidence values obtained in this study. Larger

inoculum loads were applied to both Winnipeg and Carman plots in 2012, with additional

applications in the fall of 2011 for the following year which contributed to the greater

airborne ascospore concentrations in both wheat and canola in 2012. Regardless of the

source of ascospores present in 2012, disease incidence remained lower in 2012 which is

a direct reflection of the influence of microclimate on ascospore germination in canola.

Relative humidity and leaf wetness were elevated during the flowering period during both

years, with higher values occurring in 2011; both favourable environmental conditions for

ascospores to land on canola plants and subsequently germinate and cause infection.

Ascospore release and dispersal was comparable among wheat and canola plots

however slight differences may have existed due to weather and microclimatic conditions

as well as canopy structures. In 2011 higher ascospore concentrations were noticed above

wheat canopies while in 2012 concentrations were slightly higher above canola canopies.

Stunted canola growth in 2012 as a result of warm temperatures may have provided an

open canopy allowing for ascospores to reach above canopy levels where spore traps

were located. Kane wheat used in 2011 provided similar but slightly higher yields than

133

AC Barrie wheat used in 2012; LAI also appeared noticeably larger in the field in 2011.

Microclimatic conditions under the Kane variety may have been more conducive to

sclerotia germination and ascospore release due to the increased canopy cover. Use of a

misting system in 2011 may also have had an impact on spore release from wheat and

canola canopies. Although ascospores were dispersed from within the wheat canopies,

ascospore concentrations were generally higher with proximity to canola canopies in the

bare soil. Canola field portions were larger than wheat field portions, providing a larger

inoculated area for spore dispersal. As a result of the ascospores being produced within an

adjacent wheat plot, there were no differences in ascospore concentrations or disease

incidences at different distances from the wheat inoculum source in the canola plots.

Inoculum present in the canola plots alone likely would have impacted the canola, and

any external sources of inoculum did not result in additional disease incidence according

to this study. In the case where a canola field without disease is adjacent to a wheat field

with presence or previous history of disease there would be an influence from the

inoculum in the wheat on the adjacent canola field through ascospore development and

dispersal.

Another factor impacting ascospore dispersal and gradients is wind. Predominant

wind directions changed often; therefore daily predominant winds were used to analyze

spore dispersal in each site-year. Although wind direction had no effect on ascospore

movement within the wheat strip, slight effects of wind direction on spore dispersal were

observed in the bare soil field portions due to the inoculated wheat strip in Winnipeg in

2012. This agrees with previous findings on the ascospores of L. maculans where the

134

majority of spores are carried in the direction of prevailing winds (Guo and Fernando,

2005). Wind direction affects direction of spore dispersal away from an inoculum source.

Successful disease development requires necessary weather and microclimatic

conditions as well as the presence of internally or externally produced inoculum sources.

Inoculum producing fields require conditions of elevated moisture for sclerotial

germination and ascospore release (Turkington et al., 2011). This is also true for

ascospore production among blackleg of canola and fusarium head blight of wheat (Guo

and Fernando, 2005; Government of Saskatchewan, 2007). Susceptible crops subject to

infection require adequate moisture, especially in the form of leaf wetness for successful

spore deposition and colonization (Huber and Gillespie, 1992). The major cause of

sclerotinia stem rot is through the presence of inoculum produced within the susceptible

field. In the absence of field borne inoculum, ascospores formed in adjacent fields of host

and non-host crops are capable of dispersal into susceptible fields. Control of sclerotinia

within susceptible crops is of primary concern; however canola growers should be aware

of disease history in adjacent crops. Neighbouring non-host crops can be treated using a

biological control agent applied to the soil to reduce sclerotial germination and eliminate

spore production. Crop rotation with non-host crops during several successive years can

also help deplete sclerotia in the field and reduce inoculum levels, however ascospore

production continues to occur in these crops as long as scerotia are present. Fungicides

are also advised in susceptible crops located in proximity to fields with a history of

disease. Timing of application of fungicides can be facilitated through weather

predictions focusing on precipitation and relative humidity. Additional studies conducted

in larger commercialized fields are required to determine the extent to which ascospores

135

arising from non-host crops can travel and infect neighbouring host crops and the range of

microclimatic and weather conditions required for successful infection in commercial

fields.

136

3.6 Conclusion

Disease development was not significantly altered as a result of varying

microclimates. Spore release in wheat and canola occurred during similar times indicating

that weather does have an overall influence on ascospore release from apothecia. Major

peaks followed prolonged increases in relative humidity which were generally due to

precipitation events. This finding agrees with previous literature. Misting did not

significantly alter microclimates or ascospore release under the wheat strip. Leaf wetness

was the only factor showing significantly higher values under the misting portion in 2011;

however no significant increase in ascospore concentration occurred as a result.

This study has not only proven the possibility of spore production within non-host

crops, but also that inoculum spread from neighbouring non-host crops in fields

containing inoculum from previous years is possible due to the presence of ascospores in

similar and greater amounts to those found in the adjacent canola plots. A distance of at

least 7 meters was travelled by the spores and much greater travel distances have been

reported. High levels of inoculum at 7 meters also provided evidence that the travel

distances are substantial. Timing of ascospore release under the wheat field was similar to

that of canola with peaks in ascospore release occurring during the canola flowering

period.

Wind direction analyzed against ascospore concentrations on either side of the

wheat strip showed no effect on ascospore movement within the wheat strip. Ascospore

dispersal was slightly impacted by wind direction based on the ascospore counts found

within the rotorods placed alongside the wheat strips in Winnipeg during 2012.

137

3.7 References



Boland,G.J., and Hall, R. 1988. Relationships between the spatial pattern and number of

apothecia of Sclerotinia sclerotiorum and stem rot of soybean. Plant Pathology 37:329-

336.




Clarkson, J.P., Staveley, J., Phelps, K., Young, C.S., and Whipps, J.M. 2003. Ascospore release and survival in Sclerotinia sclerotiorum. Mycol. Res. 107:213-222.


Disease.Fusarium Head Blight. Access from:


b1e2c94a03e9

Guo, X.W., and Fernando, W.G.D. 2005. Seasonal and diurnal patterns of spore

dispersal by Leptoshpaeria maculans from canola stubble in relation to environmental

conditions. Plant Disease 89:97-104.

Harthill, W.F.T. 1980. Aerobiology of Sclerotinia sclerotiorum and Botrytis cinerea

spores in New Zealand tobacco crops. New Zealand Journal of Agricultural Research

23:259-262.

Harthill, W.F.T. and Underhill, A.P. 1976. “Puffing” in Sclerotinia sclerotiorum and S.

minor. New Zealand Journal of Botany. 14(4):355-358

Huber, L. and Gillespie, T. J. 1992. Modeling leaf wetness in relation to plant disease

epidemiology. Annual Revised Phytopathology. 20:553-577.

Jamaux, I., Gelie, B. and Lamarque, C. 1995. Early stages of infection of rapeseed

petal and leaves by Sclerotinia sclerotiorum revealed by scanning election microscopy.

Plant Pathology 44(1):22-30.

Manitoba Agriculture, 2013. Crops – Diseases – Sclerotinia in Canola. Available from

http://www.gov.mb.ca/agriculture/crops/plant-diseases/sclerotinia-canola.html






138

McCartney, H.A. and Lacey, M.E. 1991. The relationship between the release of


United Kingdom. Grana 30:2, 486-492.

Mwiindilila, C.N., Hall, R. 1989. Forecasting white mold in white bean. Canadian

Journal of Plant Pathology. 11.

Qandah, I.S. 2008. Epidemiological studies on Sclerotinia stem rot caused by Sclerotinia

sclerotiorum (Lib.) de Bary on canola. Ph.D. Dissertation, North Dakota State University,

Fargo, ND.





Sclerotinia stem rot gradients in canola fields. Canadian Journal of Plant Pathology

34(3):390-400.

Turkington, T.K., Kutcher, H.R., McLaren, D., and Rashid, K.Y. 2011. Managing

Sclerotinia in oilseed and pulse crops. Prairie Soils & Crops Journal. Insects and

Diseases. Volume 4.



139

4. OVERALL SYNTHESIS

To my knowledge, this is the first report that attempts to determine the combined

impact of varying canopy densities and microclimate on both ascospore release and

infection of sclerotinia stem rot disease in Canola in Manitoba. The effects of weather on

disease development were analyzed in this study as well. Also, not previously determined

was the development, release and spread of S. sclerotiorum ascospores in a non-host

wheat crop.

Canopy density treatments effectively altered within canopy microclimates, by

increasing within-canopy temperatures and decreasing relative humidity under less dense

canopies; conditions expected to reduce disease development. Ascospore release was not

influenced by canopy density in this study. Percent infection was significantly increased

as a result of reduced temperatures and increased relative humidity among higher density

plots in Carman during 2011; however no other site-years showed similar results. Jurke

and Fernando (2008) found that disease incidence was increased as a result of denser

canopies creating favorable microclimatic conditions and more lodging. Although

increasing canopy density is useful in increasing yields and reducing weed pressures,

diseases such as sclerotinia and blackleg favor moist, humid conditions present under

denser canopies. According to this study, the below canopy microclimate modifications

may not have been sufficient to effectively alter disease development; however there is

some evidence showing otherwise especially in Jurke and Fernando‟s (2008) study. As a

precautionary measure, using average row spacing and normal seeding rates would be

140

beneficial in slightly controlling lodging and conditions conducive to infection.

Recommended rates will also guarantee yields and alleviate excess weed pressures.

Further study on the influence of canopy density on disease development is required

under various climates including drier locations that would show greater below canopy

alterations as a result of canopy density modification.

The seasonal pattern of ascospore dispersal by S. sclerotiorum under Manitoba

weather and microclimate conditions was determined in this study. Ascospore release

concentrations were higher during late bloom compared to early bloom which may be a

result of increased precipitation during the latter part of the flowering period.

Precipitation plays a key role in the onset of fungal spore release according to research

completed on fungal diseases including S. sclerotiorum, F. Graminearum and L.

Maculans (Qandah and Mendoza, 2011; Government of Saskatchewan, 2007; Guo and

Fernando, 2005). It is safe to assume that climate is a key driver for ascospore release

since peaks occurred simultaneously in Winnipeg and Carman; two locations 50 km apart

with similar climatic conditions. Concurrent with the findings of Qandah and Mendoza

(2011), this study shows that elevated ascospore concentrations seem to occur as a result

of a prolonged increase in relative humidity which occurs as a result of a precipitation

event followed by an increase in temperature and decrease in relative humidity. These

findings are not completely evident. Although precipitation influenced ascospore

concentrations, the effects of misting on ascospore release are still uncertain. Additional

focus needs to be placed on the effects of misting on ascospore release, especially in drier

regions where misting creates greater changes within a microclimate. In order to better

control sclerotinia economically, it is important to focus on several risk management

141

strategies concerning weather and climate. Forecasting weather conditions, including

precipitation, relative humidity and temperature especially just prior to and during the

onset of flowering is essential in predicting the need for fungicide applications. A similar

study using both rotorods and a burkard spore trap would aid in determining diurnal

ascospore release times which would help predict seasonal patterns focusing on a

specified period during the day.

Infection was not increased as a result of elevated ascospore concentrations at any

of the site-years indicating that the environment may have been more influential than

ascospore concentration. Increased relative humidity values and slightly reduced

temperatures experienced in Carman compared to Winnipeg may be responsible for the

higher infection in Carman. Precipitation only correlated to increased infection where

inoculum levels were even among site-years. Even sclerotia dispersal in Winnipeg and

Carman during 2012 with no previous history of disease in either field showed an impact

of increased precipitation leading to more infection. Inoculum levels most closely

correlated to infection, where fields and plots with increased inoculum levels and

previous history of disease experienced increased infection. This highlights the

importance of knowing the previous crops grown in the field as well as the history of

disease in order to better manage a crop susceptible to sclerotinia stem rot disease either

through crop rotation or fungicide use.

Under a non-host wheat canopy, sclerotia effectively produced apothecia and

released ascospores into the surrounding atmosphere. Timing and quantity of ascospore

release in wheat was similar to that in canola. Ascospores were capable of travelling up to

142

at least 7 meters beyond the inoculum source in this study and records of S. sclerotiorum

ascospore travel of at least 63 meters has been observed (Qandah and Mendoza, 2012).

Wind also played a slight role in the movement of ascospores away from the inoculum

source. Due to the ability for ascospore production, release and dispersal under non-host

crops, it is important for canola growers to recognize the impact of neighboring fields

with a history of disease even when non-host crops are grown. The recommendation by

the Canola Council of Canada (2014) to separate fields with previously grown canola

containing blackleg in the last year by 50 to 100 meters is also reasonable for sclerotinia.

Additional research needs to focus on inoculum persistence in fields containing non-host

crops. Knowlege of the annual rotation required to diminish inoculum concentrations

would be useful in helping farmers to reduce and prevent infection.

Sclerotinia forecasting models as well as risk point systems provide useful tools

for demonstrating known relationships between SSR related factors, and also help canola

growers in decision-making for better management practices. This study was not able to

further enhance these tools since ascospore release and disease incidence was not

significantly affected by the changes in microclimate under the varying canopy density

treatments. It is possible that these changes in microclimate were insufficient to impact

disease development. Since peaks in ascospore release concentrations occurred on the

same days during two consecutive years at two locations more than 50 km apart, there

appears to be a combined effect of weather and microclimatic conditions on ascospore

release which has yet to be determined.

143

In summary, the results of this study have clarified the temporal pattern of S sclerotiorum

ascospore release in canola and wheat. The influence of variable canopy densities on

microclimate, spore release and percent infection was also determined and demonstrated

that spore release and percent infection were not greatly affected by canopy density

alterations. Most importantly, it was discovered that ascospore release occurs under non-

host crops and that these ascospores are capable of infecting nearby fields containing host

crops.

144

4.1 References




Government of Saskatchewan, 2007. Home. About Agriculture Crops, Crop Protection.

Disease. Fusarium Head Blight. Access from:


b_l_e2c94a03e9

Guo, X.W., and Fernando, W. G. D. 2005. Seasonal and diurnal patterns of spore

dispersal by Leoptosphaeria maculans from canola stubble in relation to environmental

conditions. Plant Disease 89:97-104.

Jurke, C.J. and Fernando, W.G.D. 2008. Effects of seeding rate and plant density on


41(2): 142-155.





Sclerotinia stem rot gradients in canola fields. Canadian Journal of Plant Pathology

34(3):390-400.

http://www.canolacouncil.org/canola-encyclopedia/diseases/blackleg/#fnote3

http://www.canolacouncil.org/canola-encyclopedia/diseases/blackleg/#fnote3

http://www.agriculture.gov.sk.ca/Default.aspx?DN=24a3bb18-a096-4bcd-889c-b_l_e2c94a03e9

http://www.agriculture.gov.sk.ca/Default.aspx?DN=24a3bb18-a096-4bcd-889c-b_l_e2c94a03e9

145

Appendix A

Figure A.1 Sclerotinia stem rot disease life cycle including management practices in blue

indicated by the numbers 1 through 7 and environmental conditions impacting

disease development in red indicated by letters A through F.

OVERWINTERING SCLEROTIA IN THE SOIL

APOTHECIA FORMATION UNDER

THE CANOPY

ASCOSPORE RELEASE AND DISPERSAL

SPORES LAND ON PETALS DURING FLOWERING

LESIONS DEVELOPS ON PETALS

LESION PROGRESSES DOWN STEM, PLANT WILTS AND STEM BECOMES BRITTLE

NEWLY FORMED SCLEROTIA IN STEM

6

Management Practices

1) Canola seed treatment 2) Biological control 3) Tillage

4) Disease forecasting

5) Genetic resistance

6) Fungicide application

7) Crop rotation

1, 2, 3, 4, 5 7

Environmental Conditions impacting disease development

A) Temperature B) Relative Humidity C) Leaf Wetness D) Soil Moisture E) Precipitation F) Wind

A, B, D, E

A, B, E

F

C

A, B, C, E

A, B, C, E

146

Figure A.2 Canola plots containing 1 microclimate station and 1 rotorod in each plot

during flowering in Carman, Manitoba (2012).

Figure A.3 Wheat strip located adjacent to the canola plots with rotorods and

microclimate stations in Winnipeg, Manioba (2011).

147

7.

8.

9.

Figure A.4 Canola plots in Winnipeg (2011) with microclimate stations and rotorods

during the leaf stage.

148

Figure A.5 Hobo microclimate station.

149

Figure A.6 Rotorod spore sampling device.

weather, microclimate, canopy density and neighbouring non

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